Methods of identifying glycopeptides recognized by disease-associated auto-antibodies

ABSTRACT

Methods for identifying glycopeptides and more particularly glycopeptide epitopes that are specifically recognized by disease-associated auto-antibodies are provided. In some aspects the auto-antibodies are cancer-associated or autoimmune disease associated. In other aspects, methods of diagnosing a patient with cancer or an autoimmune disease, or for eliciting an immune response in a mammalian host directed to the glycopeptides of the invention are provided.

CROSS REFERENCE TO PRIOR APPLICATION

This application is a continuation of U.S. application Ser. No. 11/987,034, filed Jan. 7, 2011, which claims priority to U.S. Provisional Application Ser. No. 61/293,583, filed Jan. 8, 2010 and Ser. No 61/294,477 filed Jan. 12, 2010, all of which are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention is related to methods for identifying glycopeptides and more particularly glycopeptide epitopes that are specifically recognized by disease-associated auto-antibodies. In some aspects the auto-antibodies are cancer-associated or autoimmune disease-associated. The invention also relates to methods of diagnosing a patient with cancer or an autoimmune disease, or for eliciting an immune response in a mammalian host directed to a glycopeptide of the invention. The invention also relates to novel individual glycopeptides as well as to panels of glycopeptides (whether individually novel or not) useful for autoantibody detection purposes.

BACKGROUND OF INVENTION

Malignant transformation of cells is virtually always accompanied by alterations in the posttranslational modifications (PTMs) of proteins, and one of the best documented examples is the abundant mucin-type O-glycosylation (hereafter referred to as O-glycosylation) found on mucins and other O-glycoproteins (Tarp and Clausen 2008). Tumor-associated changes in expression of O-glycoproteins and/or in their aberrant glycosylation, create a diverse set of unusual molecular structures found on the surface of cancer cells as well as in secretions. These molecular structures generally represent glycoproteins with truncated immature O-glycans, to which the immune system of man is not normally exposed except, in some cases, as biosynthetic intermediates and then only in the secretory pathway. Therefore, these structures may represent a different form of tumor-associated antigens (one not necessarily based on differential expression level or sequence mutation), to which individuals lack immunological tolerance and thus provoke both auto-antibodies and cell mediated immunity (Anderton 2004; Doyle and Mamula 2005; Doyle and Mamula 2001).

Several mouse antibodies have been isolated and characterized as having unique binding specificity for combined glycopeptide epitopes that include both the peptide sequence as well as the aberrant PTM (hereinafter “APTM”) for efficient binding. Examples include monoclonal antibodies that specifically recognize distinct O-glycopeptides from MUC2, MUC1, and other glycoproteins (Reis et al. 1998; Sorensen et al. 2006; Danielczyk et al. 2006; Dian et al. 2009; Li et al. 2009; Takeuchi et al. 2002; Clark et al. 1998).

There exist examples of human antibodies with selective or specific reactivity with PTM-modified proteins, but these are generally limited to inflammatory and autoimmune diseases (Anderton 2004; Doyle and Mamula 2005; Doyle and Mamula 2001). Furthermore, human hydridoma technologies have identified natural IgM antibodies that react with glycoforms of proteins but the nature of the epitopes has not been fully clarified (Rauschert et al. 2008; Vollmers and Brandlein 2009; Brandlein et al. 2004a; Brandlein et al. 2004b; Rasmussen and Ditzel 2009). A major drawback of current technologies to screen for auto-antibodies directed against APTM proteins is the lack of high through-put methods for identifying antigens, more particularly epitopes, for generating pertinent antigens and for screening such antigens.

In principle, cancer-associated auto-antibodies represent appealing potential biomarkers. Auto-antibodies may develop early in carcinogenesis, at the time tumor-associated antigens appear on premalignant or malignant lesions. Antibody responses can produce relatively high concentrations in circulation with a long circulation time, and they can be detected with sensitive and specific methods (see, (Lu et al. 2008; Anderson and Labaer 2005)). In contrast, antigens produced by small premalignant or malignant lesions are generally produced in vanishingly small levels that due to dilution and clearance from blood may not be detectable by conventional techniques. Discovery and characterization of specific auto-antibodies to cancer antigens have been undertaken using different approaches in the past. Classical studies identified such antibodies reactive with tumor cells, tissues, or isolated proteins (Kawabata et al. 2007), but distinct molecular features of binding epitopes have generally not resulted from these approaches.

More recent proteome-wide screening techniques have included expressed cDNA libraries (SEREX) (Sahin et al. 1995), protein and peptide arrays (Stockert et al. 1998; Pereira-Faca et al. 2007), random or designed phage displays (Mintz et al. 2003), and more recently self-assembling protein arrays (Ramachandran et al. 2008; Anderson et al. 2008). Cancer-associated auto-antibodies characterized to date have been found to bind intracellular proteins with functions important in cell cycle regulation, such as GPR78 (Mintz et al. 2003), p53 (Lubin et al. 1993), NY-ESO-1, and CDC25 (Liu et al. 2008), but also some cell membrane glycoproteins such as MUC1 (Snijdewint et al. 1999), HER2 (Chapman et al. 2007) and Mesothelin (Hellstrom et al. 2008).

Auto-antibodies are believed to be induced as a result of altered expression of proteins and altered molecular structure due to mutations, alternative splicing and post-translational events such as protein processing and aberrant enzymatic modifications including glycosylation (Anderton 2004; Doyle and Mamula 2005; Doyle and Mamula 2001). These events induce breakage of tolerance and immunity may result. Surprisingly, however, few disease- or more specifically cancer-associated auto-antibody epitopes have been identified and molecularly defined despite considerable efforts and broad proteome screening. This is due to limitations in methods for identification of such auto-antibodies, in that, before the present invention, the appropriate antigen epitopes have not been determined and hence not tested to lead to identification of disease-associated antibodies, to serve as substrates for the detection of disease or to serve as prototype vaccines for induction of immunity against the epitopes of these autoantibodies.

There are few known examples of disease-associated human antibodies to proteins involving glycosylation. One important example is an immunodominant epitope in type II collagen comprising a glycosylated hydroxylysine residue that is involved in collagen-induced arthritis (Backlund et al. 2002). Glycosylation may also modulate protein processing and hence affect exposure of new epitopes as shown in Rasmussen's encephalitis, where an N-glycan blocks proteolysis of a neuronal glutamate receptor and a short preceding peptide epitope (Gahring et al. 2001). Several human monoclonal antibodies have been shown to be directed to epitopes affected by glycosylation (Rauschert et al. 2008; Vollmers and Brandlein 2009; Brandlein et al. 2004a; Brandlein et al. 2004b; Rasmussen and Ditzel 2009), but the nature of the molecular epitopes remains undefined.

There is therefore a need to develop methods for the identification of PTM-containing peptides, such as aberrant glycopeptides (hereinafter “AGP”), that are specifically recognized by disease-associated auto-antibodies. The present invention provides such methods. There is also a need for improved diagnostic tools, such as AGP, that would permit early detection of disease, notably cancer, for example by being used as substrates to capture disease-associated autoantibodies.

SUMMARY OF INVENTION

In one embodiment, the invention provides a method for identifying glycopeptides reactive with cancer-associated auto-antibodies, the method comprising: providing a panel comprising peptides, at least a plurality of the peptides comprising one or more sites amenable to glycosylation, wherein at least one of the glycosylation sites has been modified with a glycan to form a glycopeptide having a peptide portion and a glycan portion; contacting the panel with an antibody-containing sample from a patient with cancer; and identifying glycopeptides in the panel that (i) are selectively recognized by antibodies in the sample, but not by antibodies in a control sample and (ii) are recognized by such antibodies in the sample through recognition of both the peptide portion and the glycan portion and not through recognition of either the peptide or the glycan alone.

In another embodiment, a panel of the invention comprises a plurality of peptides having an amino acid sequence comprising at least one serine or threonine residue, wherein the residue is a glycosylation site. In yet another embodiment, the at least one serine or threonine residue is at about the middle of the amino acid sequence. In still another embodiment, a panel of the invention comprises mutants of the peptides. The number of peptides in a panel may vary and may be at least 8 peptides, for example between about 8 and about 30 peptides, such as 10, 12, 15 or 20 peptides. A number of peptides in excess of 30 is also within the invention. The upper limit of peptides in a panel is limited by practical considerations (e.g., how many peptides can fit on a substrate) or cost-benefit considerations. Preferably the peptides are selected from the group consisting of SEQ ID NOs 15, 36, 49, and 82-146.

In a specific aspect of the above embodiment, providing a method for identifying glycopeptides reactive with cancer-associated auto-antibodies, the method further comprises identifying cancer-associated glycopeptide epitopes. In another aspect, the method further comprising elucidating the epitope structure of the identified glycopeptide epitopes.

In a specific embodiment, the invention provides a method for determining whether a patient has cancer, the method comprising: contacting an antibody-containing sample from the patient with a panel comprising peptides at least a plurality of which are glycopeptides, each glycopeptide comprising a glycopeptide epitope, the epitope having been previously determined (i) to be selectively recognized by a subset of antibodies in sera from cancer patients, which subset recognizes neither (a) the corresponding naked peptides of the panel when not glycosylated; nor (b) the corresponding glycan when not bound to the peptide; and (ii) not to be recognized by antibodies in control sera; contacting the panel with an antibody-containing sample from the patient; determining if antibodies in the sample are bound to glycopeptides of the panel; and concluding either that the patient has cancer if the sample comprises antibodies that bind to at least one of the glycopeptides in the panel; or that the patient does not have cancer if the sample does not comprise antibodies that bind to at least one glycopeptide in the panel.

In one aspect of the above embodiment providing a method for determining whether a patient has cancer, the patient is newly diagnosed with cancer. In another aspect, the method further comprises concluding either that the patient has cancer if the sample comprises IgG antibodies that bind to at least one of the glycopeptides in the panel; or that the patient does not have cancer if the sample does not comprise IgG antibodies that bind to at least one glycopeptide in the panel. In yet another aspect, the patient is diagnosed with cancer if said sample comprises antibodies that specifically recognize one or more of the glycopeptides having an amino acid sequence selected from the group consisting of: SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146).

In another embodiment, the invention provides a method for eliciting an immune response in a patient, the method comprising administering to the patient a composition comprising (i) a glycopeptide consisting essentially of an amino acid sequence selected from the group consisting of SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146), in an effective amount for eliciting the immune response; and (ii) a suitable adjuvant.

In a specific aspect of the above embodiment, providing a method for eliciting an immune response in a patient, the immune response comprises an IgG antibody response. In another aspect, the immune response is an anti-cancer immune response. In yet another aspect, the cancer is selected from the group consisting of breast cancer; colon cancer; ovarian cancer; cervical cancer; pancreatic cancer; prostatic cancer; liver cancer; kidney cancer; brain cancer; hematological cancer; testis cancer; head and neck cancer; and lung cancer.

In one aspect of the above embodiment, providing a method for eliciting an immune response in a patient, the glycopeptide is modified with an O-glycan at at least one amino acid residue. In another aspect, the at least one amino acid residue is a serine or threonine residue.

In any of the above embodiments of the invention, each of the peptides in the panel may be about 2 to about 50 amino acid residues in length. In other aspects, each of the peptides in the panel may be about 4 to about 25 amino acid residues in length.

In certain aspects of the above embodiments of the invention, the amino acid sequence of the peptides of the panel is a fragment found in a protein or variant of said protein or a conservative mutant. In some of the above embodiments, the identified glycopeptides of the panel are found in at least one glycoprotein that is aberrantly glycosylated in cancer cells. In yet other of the above embodiments, glycopeptides identified by the methods of the present invention are found in at least one glycoprotein that is overexpressed in cancer cells.

In other aspects, the glycopeptides are synthesized synthetically or chemoenzymatically. In still other of the above aspects, the glycopeptides of the panel are partially glycosylated peptides when immobilized on the panel.

In certain of the above embodiments, the control sample contains pooled samples from a plurality of control individuals.

In certain of the above aspects of the invention, the panel is a microarray slide. In other aspects, the glycopeptides identified by the methods of the invention are selectively recognized by an IgG antibody. In certain of the above embodiments, the panel comprises one or more glycopeptides having an amino acid sequence selected from the group consisting of: SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146).

In another aspect, the invention provides a glycopeptide comprising an amino acid sequence selected from the group consisting of SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146).

In yet another aspect, the invention provides a glycopeptide consisting essentially of an amino acid sequence selected from the group consisting of SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146).

In still another aspect, the invention provides a glycopeptide consisting of an amino acid sequence selected from the group consisting of SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49), LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146).

In another aspect, the invention provides a pharmaceutical composition comprising one or more glycopeptides selected from the group consisting of SEQ ID NOs 15, 36, 49, and 82-146. Preferably, the pharmaceutical composition comprises one or more glycopeptides selected from the group consisting of LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146).

In yet another aspect, the invention provides for a panel of glycopeptides comprising at least a plurality of glycopeptides, each glycopeptide comprising a glycopeptide epitope, said epitope having been previously determined (i) to be selectively recognized by a subset of antibodies in sera from cancer patients, which subset recognizes neither (a) the corresponding naked peptides of said panel when not glycosylated; nor (b) the corresponding glycan when not bound to said peptide; and (ii) not to be recognized by antibodies in control sera, said plurality comprising at least 8 glycopeptides selected from the group consisting of glycopeptides having SEQ IDs 15, 36, 49, and 82-146. Preferably, the plurality of glycopeptides comprises SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146).

In any of the above embodiments of the invention, the glycopeptides of the panel may be glycosylated in situ, in solution, or in vivo by recombinant expression in a host cell. In any of the above embodiments, the glycopeptides of the panel may be treated with one or more exoglycosidases to expose O-glycans. In still other of the above embodiments, the glycan may be an O-glycan. In any of the above embodiments, the O-glycan may be a member selected from the group consisting of: Tn, STn, T, Truncated C3, Truncated C2, Truncated C4, non-capped type1-C3, non-capped type2-C2, non-capped type2-C4, GalNAca-Tn, SA-type1-C3, SLea-C3, LacDiNAc-C3, LacDiNAc-C2, and LacDiNAc-C4.

In certain of the above embodiments, at least one glycopeptide in the panel is not a glycopeptide comprising a glycosylated GSTA motif.

In certain of the above embodiments, none of the glycopeptides in the panel are glycopeptides comprising a glycosylated GSTA motif.

These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an image of a microarray slide and illustrates an example of on-slide (in vitro) glycosylation with a polypeptide GalNAc-transferase, GalNAc-T3, to glycosylate peptides and GalNAc glycopeptides with additional unsubstituted Ser/Thr residues to enhance number of O-glycans. FIG. 1A: GalNAc-T3 untreated array, FIG. 1B: GalNAc-T3 treated array.

FIG. 2 is an image of a microarray slide and illustrates reactivities of serum (1:25 dilution) from a newly diagnosed prostate cancer patient (#762 in FIG. 2A) and a normal control serum from an individual that does not have prostate cancer (#174 in FIG. 2B) on the library of 96 paired peptides/GalNAc-glycopeptides as designated in Table II.

FIGS. 3-5 are bar graphs and illustrate microarray screening results from 147 cancer and 31 control sera on the glycopeptide pairs identified herein as #20/21 (FIG. 3), #62/63 (FIG. 4), or #86/87 (FIG. 5).

FIG. 6A-C are bar graphs showing 8 selected glycopeptides (i.e., 889, 275a, 585, 893, 931-C3, 852-C3, 873, and 690) reactive with IgG from newly diagnosed cancer patients compared to normal control sera. FIG. 6A: glycopeptides 889 (SEQ ID NO: 134), 275a (SEQ ID NO: 86), and 585 (SEQ ID NO: 109). FIG. 6B: glycopeptides 893 (SEQ ID NO: 135), 931-C3 (SEQ ID NO: 146), and 852-C3 (SEQ ID NO: 145). FIG. 6C: gycopeptides 873 (SEQ ID NO:132) and 690 (SEQ ID NO: 116). The following human sera were used for FIGS. 6A and 6B: 32 ovarian cancer (O), 38 breast cancer (B), 54 colon cancer (C), 17 lung cancer (L), as well as 145 normal sera (N) (Asterand Corp). The following sera were used for FIG. 6C: 32 ovarian cancer (O), 38 breast cancer (O), 54 colon cancer (C), 42 lung cancer (L), 52 pancreatic cancer (Pn), 35 prostatic cancer (Pr), 8 inflammatory disease (In), 8 benign controls (Bn), and 145 healthy controls (N). Sera were screened at 1:20 dilution. Anti-human-IgG conjugated to Cy3 was used as the secondary detection unit and slides were scanned with a GenePix 4200AL Microarray Scanner.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for identification of peptides and peptide epitopes that comprise one or more posttranslational modification(s) (PTM(s)) (“PTM-peptides”/“PTM peptide epitopes”) that are selectively recognized and bound by human disease-associated antibodies. These will include peptides that display an aberrant glycosylation pattern either due to an alteration in sequence (mutation) which invites a changed glycosylation pattern, or simply a glycosylation pattern not normally encountered in cancer-free individuals. Such peptides will constitute AGP as the term is introduced and hence defined above. For the removal of doubt, PTM-epitopes are not whole molecules; therefore, any discussion of such epitopes should be construed as applying to glycopeptides harboring such epitopes.

In some aspects, the PTM-peptides of the invention are useful for diagnosing a disease. In a specific embodiment, the present invention provides methods for diagnosing a patient with cancer. In another embodiment, the patient is newly diagnosed with cancer (i.e., diagnosed with cancer for the first time). In still other embodiments, a patient is diagnosed with an autoimmune disease.

In certain embodiments, the methods are useful for identifying PTM-peptides that are useful for eliciting in an individual an immune response directed against PTM-epitopes that are associated with disease, such as cancer. In certain embodiments, the immune response includes an auto-antibody response. In certain aspects, the induction of auto-antibodies to aberrant PTM-modified glycoproteins includes antibodies that specifically recognize O-glycopeptide epitopes.

As discussed, supra, malignant transformation of cells is virtually always accompanied by alterations in the posttranslational modifications (PTMs) of proteins, including O-glycosylation (Tarp and Clausen 2008). Tumor-associated changes in expression of O-glycoproteins and/or in their aberrant glycosylation, create a diverse set of unusual molecular structures found on the surface of cancer cells as well as in secretions. These molecular structures generally represent glycoproteins with truncated immature O-glycans, to which the immune system of man is not normally exposed except, in some cases, as biosynthetic intermediates and then only in the secretory pathway. Thus, one aspect of the PTM-peptides and PTM peptide epitopes identified by the methods of the present invention, i.e., AGP, is that they are not covered by immunological tolerance, and hence are potential targets for immunotherapeutic intervention. The aim of eliciting immune responses in a host using the AGP of the invention is the development of vaccines against the very disease that the AGP or other PTM-peptides are associated with.

Thus, AGP can cause induction of auto-antibodies to PTM-peptide epitopes. One aspect of this invention is the design and production of PTM-peptide libraries that represent or more accurately contain AGP associated with disease. In another aspect of the invention, high through-put methods for screening of disease-associated antibodies using such libraries are provided. In a specific embodiment, the invention relates to aberrant O-glycosylation of glycoproteins and more specifically, of glycopeptides. The present inventors believe one of the reasons why these disease-associated PTM-peptides and epitopes have eluded detection and especially association with disease is that the proteins bearing them in vivo are not or not necessarily mutated or overexpressed and hence would not be detected by ordinary, nontargeted, techniques.

PTM modifications can be the result of the addition of chemical groups to a protein, such as a phosphate group or a sugar moiety (e.g. acetylation of lysine and serine, glycosylation of asparagine, serine, threonine, hydroxyl-proline, lysine, methylation of arginine, histidine, and lysine, phosphorylation of serine, threonine, and tyrosine). They can also be the result of a conversion of an amino acid to a distinct structure, as in the deimination of arginine to citrulline or the deamidation of aspartic acid/asparagine to isoaspartic acid. While this invention is primarily concerned with glycosylation, it is evident that other aberrant posttranslational modifications as described herein may result in altered proteins harboring aberrant PTM-epitopes that may be the target of auto-immunity.

In some aspects of the present invention, the discriminating characteristic of the peptides and peptide epitopes useful in the present invention is that disease-associated antibodies bind selectively with the PTM-containing peptide and not with the same unmodified peptide or the PTM in the context of a different peptide sequence or another unrelated (e.g., artificial) carrier. In terms of AGP having diagnostic and/or immunogenic value, the disease-associated autoantibodies should recognize only the combination of the relevant glycosylated amino acid sequence and not the same glycan on a different peptide nor the same amino acid sequence bearing a different glycan. This will avoid false positives. It will be understood that the construction of peptide libraries and the availability of disease (e.g., cancer) and control sera that can be tested renders it unnecessary to know beforehand which AGP will be recognized by disease-associated antibodies in order to identify these antibodies. Once the disease-associated antibodies have been identified, they, or man-made versions thereof can be used to pinpoint the AGP that pulled relevant autoantibodies out of the sera.

In other aspects, the invention provides methods for the diagnosis of a patient with a disease, such as, e.g., cancer, wherein detection of antibodies in a sample from the patient that selectively recognize one or more combinations of disease-associated PTM-containing peptides is used to predict and diagnose disease. In still other embodiments, methods for treating a patient with cancer using compositions or vaccines comprising PTM-containing peptides or proteins of the invention are provided.

DEFINITIONS

As used herein, the term “immune response” includes an adaptive immune response, including a T cell response and B cell response. Thus, as used herein, the term “eliciting an immune response” means that an adaptive immune response is induced by administration of an appropriate immunogen (antigen or antigen plus carrier).

As used herein, an “anti-cancer immune response” is an immune response that is directed toward cancer cells or products secreted or shed from cancer cells. For example, an anti-cancer immune response may be characterized by tumor-specific antibodies and/or cytotoxic T cells that attack cancer cells or react specifically with a glycoprotein shed from cancer cells.

An “auto-antibody” is an antibody that specifically recognizes an epitope harbored by a self-product such as a protein, carbohydrate or lipid produced by healthy or diseased cells of an individual.

As used herein, the terms “selective binding” and “selective recognition” and their grammatical variants, of an epitope by an antibody means that the antibody binds with significantly greater affinity to the epitope compared to any other sequence or structure.

The term “epitope” refers to the part of an antigen that is specifically recognized and bound by an antibody. The term “glycopeptide epitope” is an epitope that includes both part of a peptide sequence and at least part of a glycan. The term “minimal epitope” or “minimal glycopeptide epitope” refers to the shortest glycopeptide that is recognized by antibodies recognizing the same epitope.

As used herein, the term “corresponding peptide” refers to the unglycosylated form of a glycopeptide of the invention. Thus, a glycopeptide and its corresponding peptide have the same amino acid sequence.

The term “glycan hapten” refers to glycan moiety independent of whether this is conjugated to a carrier, such as a peptide or protein (i.e. binding to the glycan hapten does not depend on the carrier such as protein or peptide to which the glycan is bound).

The terms “linear peptide” and “linear peptide epitope” means that the peptide or peptide epitope is non-conformational (i.e., antibody recognition and binding to the epitope does not depend upon the three-dimensional structure of the peptide).

The term “disease-associated antibody” means an antibody that is detected in a patient sample if the patient has the disease, but is not detected, or is present at significantly reduced levels compared to the patient sample, in a control sample. For example, a cancer-associated antibody is present in an antibody-containing sample from a patient with cancer, but is not present, or is present at significantly reduced levels in a sample from a patient without cancer. A useful distinguishing range of antibody levels is at least two or preferably three 3 fold higher levels in patients compared to healthy control. The term may also apply to groups of patients with a disease, where “disease-associated antibody” means an antibody that is detected with higher prevalence/incidence in the disease group compared to a healthy control group. A useful distinguishing prevalence (=specificity of assay) would be at least 70% and preferably 80 or 90%,

The term “aberrant glycosylation” means a glycoform that is not normally present on proteins expressed on the cell surface or secreted/shed from cells. An example of an aberrant glycoform is Tn (GalNAcα1-O-Ser/Thr), sialosyl-Tn (NeuAcα2-8GalNAcα1-O-Ser/Thr), and T (Galβ1-3GalNAcα1-O-Ser/Thr).

As used herein, a “glycosylatable peptide” or a peptide containing a “site amenable to glycosylation” refers to a peptide containing an amino acid residue that can be glycosylated. An example of a site amenable to glycosylation is a peptide site containing a serine or threonine amino acid residue, which can be O-glycosylated with N-acetyl-galactosamine (GalNAc).

As used herein, “variant” in addition to its understood meaning as a term of art includes any changes in a molecule from its wild-type form. For example, alleles, fragments, mutations, substitutions with natural or analog compounds, splice variants, glycosylations, species variants, and the like. The term is not limited to any one type of change or deviation from the wild type form or “normal” molecule. A “variant” also includes a polypeptide or enzyme which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%, and still more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared.

“Conservative mutants” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine. Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide.

An “organ-specific glycoprotein” is a glycoprotein which is only or which has substantial preferential expressed in a specific organ type. For example, the protein mucin 16 (MUC16) (SEQ ID NO: 57), is only expressed in epithelium of the female tract such as ovary and endometrium, and it is primarily overexpressed in tumors originating from these epithelia.

The term “clinical debut” refers to the first time a patient is diagnosed with a disease.

The term “immunogenic substitution” means the replacement of an amino acid residue with a different amino acid that causes the antigen to become immunogenic. For example, a substitution of an amino acid in a self protein, such as that which can result from a cancer mutation, or experimentally in a peptide, that renders the self protein immunogenic (i.e., breaks immunogenic tolerance to the self protein) is an immunogenic substitution. As another example, the substitution of a normally unmodified amino acid with a modified amino acid, wherein the modification itself (i.e., as a hapten, such as, e.g., Tn) is immunogenic, can render a protein immunogenic.

The term “subject” or “individual” as used herein refers to an animal having an immune system, preferably a mammal (e.g., rodent, such as mouse). In particular, the term encompasses humans.

As used herein, the term “about” or “approximately” usually means within an acceptable error range for the type of value and method of measurement. For example, it can mean within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.

As used herein, the term “consisting essentially of” means that the glycopeptide sequence is not limited to a length of 20 amino acids, but can be longer or shorter. For example, a glycopeptide can be less than or more than 20 amino acids long as established during the course of optimizing the length of the peptide and efficient positioning of the glycosylated epitope that is specifically recognized by auto-antibodies in patients. Throughout the specification, the length of 20 amino acids is used as a convenient length and to ensure that the entire epitope is encompassed within the peptide. While most epitopes will be on the order of 5-10 amino acid residues, and hence contained within a 20 amino acid peptide, any size peptide is possible if sufficient to contain the epitope for antibody detection. In turn, longer peptides may allow for incorporation of additional epitopes which could provide different selectivity for cancer.

Post-Translational Modification of Peptides

In some aspects, the invention relates to the identification of disease-associated PTM-peptides, wherein the PTM-peptide is selectively recognized by disease-associated antibodies. Preferably, the disease-associated antibodies recognize neither the peptide alone nor the PTM alone or attached to a different peptide. In other words, the antibody epitope comprises both part of the protein backbone and part of the PTM.

The PTM may be one or more modifications of one or more residues of the amino acid sequence. The PTM can be any of the known PTMs involving enzymatic modifications of amino acids including glycosylation, phosphorylation, citrinylation, acetylation, methylation (Anderton 2004; Doyle and Mamula 2005; Doyle and Mamula 2001).

All 20 primary amino acids used by man are capable of undergoing some type of PTM. However, certain factors determine whether those modifications will take place. First, the location of the amino acid within the protein sequence affects both the type and frequency of modifications that may arise. Flanking residues can influence the conformation of the protein, potentially altering whether an enzyme has access to a certain amino acid or is exposed to a certain environment. The cellular location of the modifying enzyme, if required, will determine whether the modification occurs and disease-associated changes in localization of such enzymes may lead to aberrant posttranslational modifications. Previous modifications or proteolytic cleavages within a protein influence subsequent amino acid modifications within the same protein.

An exemplary PTM of the invention is mucin-type O-linked glycosylation (“O-glycosylation” or “O-linked glycosylation”). Examples of O-linked glycosylation include, e.g., the addition of N-acetyl-galactosamine (GalNAc), fucose, glucose, N-acetylglucosamine (GlcNAc), or mannose. GalNAc O-glycosylation is a particularly preferred PTM of the invention. GalNAc O-glycosylation is carried out by specific enzymes; for example, the addition of N-acetyl-galactosamine (GalNAc) to serine or threonine residues is carried out by the enzyme UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (EC 2.4.1.41). Other types of O-glycosylation include αMannose, αFucose, βGlucose, βGlcNAc, and βXylose glycosylation. Also contemplated by the present invention are other forms of glycosylation, such as, e.g., N-linked glycosylation, hydroxyl-lysine glycosylation, and C-mannosylation. Biosynthesis and structures of these types of protein glycosylation are reviewed in Essentials of Glycobiology (2^(nd) edition, eds A. Varki, Cummings, Esko, Freeze, Stanley Bertozzi, Hart, Etzler. CSH Press 2009). It is to be understood that examples of PTM modification provided herein, such as O-glycosylation, are meant to be non-limiting examples which serve to prove the principal of the present methods (i.e., that the presently disclosed methods are useful for identifying PTM-modified peptides or peptide epitopes specifically recognized by disease-associated antibodies). The methods of the invention are also applicable, however, to proteins or peptides modified with other PTMs contemplated by the present invention, such as but not limited to phosphorylation, citrinylation, acetylation, and methylation.

In certain aspects of the invention, glycoproteins, glycopeptides and glycopeptide epitopes that are aberrantly glycosylated are provided. Examples of aberrant forms of O-glycans include, but are not limited to, truncated immature O-glycans such as Tn, T, and STn as well as non-sialylated and non-galactosylated core 2, 3 and 4 structures, shown in Table I below:

TABLE I Representative mucin-type O-glycan structures on normal and diseased cells¹ Type of Distribution Name Hapten Structure (+/−Ser/Thr or artificial linker) Glycoconjugate (main) No Tn GalNAcα1-O-Ser/Thr O-linked Cancer cells 1 STn NeuAcα2-3GalNAcα1-O-Ser/Thr O-linked Cancer cells 2 T Galβ1-3GalNAcα1-O-Ser/Thr O-linked Cancer cells 3 mSTa NeuAcA2-3Galβ1-3GalNAcα1-O-Ser/Thr O-linked Normal cells 4 mSTb NeuAcA2-3Galβ1-3[NeuAcα2-6]GalNAcα1-O- O-linked Normal cells 5 Ser/Thr Truncated C3 GlcNAcβ1-3GalNAcα1-O-Ser/Thr O-linked Cancer cells 6 Truncated C2 (NeuAc2-3)_(+/−)Galβ1-3[GlcNAcβ1-6]GalNAcα1-O- O-linked Cancer cells 7 Ser/Thr Truncated C4 GlcNAcβ1-3[GlcNAcβ1-6]GalNAcα1-O-Ser/Thr O-linked Cancer cells 8 Non-capped type1-C3 Galβ1-3GlcNAcβ1-3GalNAcα1-O-Ser/Thr O-linked Cancer cells 9 Non-capped type2-C2 (NeuAc2-3)_(+/−)Galβ1-3[Galβ1-4GlcNAcβ1- O-linked Cancer cells 10 6]GalNAcα1-O-Ser/Thr Non-capped type2-C4 (Galβ1-4)GlcNAcβ1-3[Galβ1-4GlcNAcβ1- O-linked Cancer cells 11 6]GalNAcα1-O-Ser/Thr GalNAcα-Tn GalNAcα1-3GalNAcα1-O-Ser/Thr O-linked Cancer cells 12 SA-type1-C3 NeuAcα2-3Galβ1-3GlcNAcβ1-3GalNAcα1-O- O-linked Cancer cells 13 Ser/Thr SLe^(a)-C3 NeuAcα2-3Galβ1-3[Fucα1-4]GlcNAcβ1- O-linked Cancer cells 14 3GalNAcα1-O-Ser/Thr LacDiNAc-C3 GalNAcβ1-3/4GlcNAcβ1-3GalNAcα1-O-Ser/Thr O-linked unknown 15 LacDiNAc-C2 Galβ1-3[GalNAcβ1-3/4GlcNAcβ1-6]GalNAcα1-O- O-linked unknown 16 Ser/Thr LacDiNAc-C4 GalNAcβ1-3/4GlcNAcβ1-3[GalNAcβ1- O-linked unknown 17 3/4GlcNAcβ1-6]GalNAcα1-O-Ser/Thr ¹Additional modifications such as capping terminal β3/4Gal residues with Fucα1-2 (blood group H) and Gal(NAc)α1-3 (blood group A,B) are generally produced by normal cells. Modifications such as 3/6-O—SO₃ of Gal and GalNAc, 9-O-acetylation of NeuAc, NeuGc instead of NeuAc, may also occur in normal and cancer cells.

In some aspects of the invention, the glycopeptide sequences of the invention may be derived from disease-associated proteins, such as, e.g., a glycoprotein specifically expressed on the cell surface of a cancer cell, but not on the surface of a non-malignant cell, or overexpressed on cancer cell surfaces, e.g., mucin 1 (MUC1) (GenBank Accession Nos. NP_002447 (SEQ ID NO: 1); NP_001018016 SEQ ID NO: 2); NP_001018017 (SEQ ID NO: 3); NP_001037855 (SEQ ID NO: 54); NP_001037856 (SEQ ID NO: 55); and NP_001037857 (SEQ ID NO: 56)); MUC16 (GenBank Accession No. NP_078966 (SEQ ID NO: 57); and mesothelin (MSLN) (GenBank Accession Nos. NP_037536 (SEQ ID NO: 58) and NP_005814 (SEQ ID NO: 59). In some embodiments, the glycoproteins are organ-specific glycoproteins. Examples of such glycoproteins include MUC16 (expressed in the female reproductive tract) and mesothelin, which is specifically expressed in the normal lung, but with cancer expression in ovarian and cervical cancers. Specific examples of other glycoproteins of the invention include, but are not limited to, human proteins predicted from the completed human genome containing one or more serine and/or threonine GalNAc O-glycosylation sites as predicted by the NetOGlyc algorithm.

Non-limiting examples of such proteins include cell membrane receptors such as low-density lipoprotein receptor precursor (LDLR) (GenBank Accesion No. NP_000518) (SEQ ID NO: 67)), very low-density lipoprotein receptor precursor (VLDLR) (GenBank Accession Nos. NP_001018066 (SEQ ID NO: 68) and NP_003374 (SEQ ID NO: 69)), and receptor tyrosine-protein kinase erbB-2 precursor (ERBB2) (GenBank Accession Nos. NP_004439 (SEQ ID NO: 70) and NP_001005862 (SEQ ID NO: 71)); chaperones such as heat shock 70 kDa protein 5 (HSPA5) (GenBank Accession No. NP_005338 (SEQ ID NO: 72) and heat shock 70 kDa protein 8 (HSPA8) (GenBank Accession No. NP_694881) (SEQ ID NO: 73) and NP_006588 (SEQ ID NO: 74); secreted cytokines, growth factors and glycoproteins such as interferon alpha-2 precursor (IFNA2) (GenBank Accession No. NP_000596) (SEQ ID NO: 75), interleukin-2 (IL2) (GenBank Accession No. NP_000577) (SEQ ID NO: 76), and inhibin beta B chain precursor (INHBB) (GenBank Accession No. NP_002184) (SEQ ID NO: 77); proteases such as matrix metalloproteinase-14 precursor (MMP14) (GenBank Accession No. NP_004986) (SEQ ID NO: 78); enzymes such as prostatic acid phosphatase precursor (ACPP) (GenBank Accession Noa. NP_001127666) (SEQ ID NO: 79) and NP_001090 (SEQ ID NO: 80); and mucins such as melanoma cell adhesion molecule (MCAM) (GenBank Accession No. NP_006491) (SEQ ID NO: 81).

In other embodiments, glycopeptide mutants are contemplated for use in the present invention. For example, a peptide library can be generated wherein each amino acid in the peptide sequence around one or more sites for PTM modification (e.g., O-glycosylation) is varied by any combination of the 20 amino acids used in human proteins to form additional peptide sequences (i.e. mutants) with the same PTM motif.

In certain embodiments, the present invention further relates to the design of glycopeptide libraries for screening disease-associated antibodies and/or for identifying disease-associated glycopeptides and/or glycopeptide epitopes. In certain aspects, a glycopeptide library of the invention is provided as a glycopeptide panel for identifying glycopeptides reactive with disease-associated auto-antibodies. A “glycopeptide panel” comprises at least two glycopeptides (or indeed entire glycoproteins) and may be used in an antibody binding assay of the invention. In certain aspects, methods for designing both the sequence of the glycopeptide and the specific site(s) for glycosylation are provided.

Preferably, glycopeptides of the glycopeptide libraries of the invention are from about 4 to about 50 amino acids in length. More preferably, such glycopeptides are from about 4 to about 25 amino acid residues in length.

In certain aspects, peptides are designed as fragments of a glycoprotein. A random peptide library encompassing peptides having between about 4 and about 50 amino acids, is provided, wherein each amino acid in each peptide is varied by all 20 amino acids used in human proteins to form different peptide sequences. These random peptides are PTM-modified, e.g., O-glycosylated, on at least one amino acid residue. In other aspects, the peptide library comprises peptides covering all serine and threonine residues in the predicted human protein sequences. In yet another aspect, peptides of the glycopeptide libraries comprise sequences derived from disease-associated proteins and/or organ-specific proteins, as described, supra.

In certain embodiments of the invention, a peptide suitable for testing is a potential acceptor of GalNAc O-glycosylation by polypeptide GalNAc-transferases if: 1) the peptide is a sequence derived from the human proteome, wherein the human proteome may be predicted from the human genome; and 2) the peptide comprises one or more serine and/or threonine residues. Preferably, such peptides are derived from proteins that have a signal sequence. It is also preferred that such peptides have sequences that, in vitro, serve as substrates for one or more human polypeptide GalNAc-transferases, such as, e.g., GalNAc-T1, T2, T3, or T4. The initiating enzymes for other types of O-glycosylation that attaches the first sugar residue to Serine and Threonine residues of polypeptides have been cloned and expressed recombinantly and hence these types of modifications are amiable to the same strategy for testing (Essentials of Glycobiology (2^(nd) edition, eds A. Varki, Cummings, Esko, Freeze, Stanley Bertozzi, Hart, Etzler. CSH Press 2009).

In certain aspects, it is preferred that the glycopeptides of the invention comprise sequences predicted by the NetOGlyc algorithm. The NetOGlyc algorithm has been developed to predict sites and proteins modified by O-glycosylation and this has been postulated to have a positive prediction rate of 76% (Julenius et al. 2005).

In other embodiments, peptides may be modified with other PTM modifications.

Glycopeptide Synthesis

In some aspects of the invention, the peptide sequence of the glycopeptides of the invention can be synthesized using standard chemical synthesis [Meldal M, Bock K. A general approach to the synthesis of O- and N-linked glycopeptides. Glycoconj J. 1994; 11(2):59-63]. For example, GalNAca-Ser/Thr-Fmoc, GlcNAcα-Ser/Thr-Fmoc, Manα-Ser/Thr, and Glcβ-Ser/Thr amino acids are commercially available (Sussex Inc (Canada)), and may be incorporated into the peptides during synthesis. The glycopeptides of the invention may be synthesized with and without an N-terminal linker for printing on epoxy or NHS-activated glass slides, respectively. In certain embodiments, glycopeptides may also be synthesized directly on microarray slides (“spot synthesis”). In other embodiments, peptides are previously synthesized and then immobilized or used in solution, according to a method of the present invention.

In certain aspects, the synthesized peptides or glycopeptides are O-glycosylated or further O-glycosylated using enzymatic synthesis of O-glycans at specific sites to form glycopeptides (Tarp and Clausen 2008).

In certain aspects of the present invention, the peptides may be glycosylated by a number of different methods, such as e.g., on-slide glycosylation, in solution glycosylation, or in vivo, e.g., by recombinant expression in appropriate host cells (Tarp et al. Glycobiology 2007).

Glycosylation may be achieved using one or more recombinant glycosyltransferases. such as, e.g., recombinant polypeptide GalNAc-transferases (e.g. GalNAc-T2, -T3 and -T4). See, e.g., U.S. Pat. No. 5,876,716 by Hansen and U.S. Pat. No. 6,465,220 by Hassan; see also, Bennett et al. 1998; Bennett et al. 1996; and White et al. 1995.

Further expansion may also be achieved using a recombinant sialyltransferase, ST6GalNAc-II, to produce STn glycoforms (structure no. 2, Table I) of the GalNAc glycopeptides. T glycoforms may be produced by a recombinant Drosophila core1 β3galactosyltransferase, truncated core3 glycoforms (structure no. 6, Table I) may be produced using a recombinant human β3GlcNAc-transferase (Iwai et al. 2002), and non-capped type1-core3 glycoforms (structure no. 9, Table I) may be produced using β3Gal-T5 (see, U.S. Pat. No. 7,332,279).

In other aspects, the glycopeptides and/or glycoproteins of the invention may be derived from recombinant or isolated glycopeptides or glycoproteins and further glycosylated or modified according to the methods of the present invention. For example, commercially available glycoprotein arrays (available, e.g., from Invitrogen), may be treated with exoglycosidases, e.g., neuraminidase, βgalactosidase, βN-acetylglucosaminidase and other enzymes, in order to expose cancer-associated glycans, e.g., Tn or T, to form glycopeptides according to the present invention.

In certain aspects of the invention, the O-glycan is preferably positioned in the center of the peptide. While not intending to be bound by a specific theory, positioning the O-glycan in the center of the peptide may facilitate proper presentation of the O-glycan for specific antibody binding to a glycopeptides epitope. In certain aspects, for example, in a glycopeptide of 20 amino acid residues in length, the O-glycan is preferably attached to a serine or threonine amino acid residue placed at a site from about residue 6 to about residue 15 and more preferably from about residue 8 to about residue 13. For example, in the present Examples, glycopeptides were designed with a single GalNAc at position 12 (of the 20-mer) to allow optimal exposure of peptide sequence flanking the O-glycan, taking into consideration that the glycopeptides will be covalently linked primarily through the N-terminal amino acid.

In certain aspects, the O-glycans to be presented on a glycopeptide library of the invention may include all known O-glycan structures. In preferred embodiments, the glycopeptides comprise glycans known to be disease-associated, such as Tn, STn, T, Truncated C3, Truncated C2, Truncated C4, non-capped type1-C3, non-capped type2-C2, non-capped type2-C4, GalNAca-Tn, SA-type1-C3, SLea-C3, LacDiNAc-C3, LacDiNAc-C2, and LacDiNAc-C4 (see Table I for additional non-limiting examples of such glycans). Further the O-glycans may be substituted with sulfation or other immunogenic substitutions including acetylation and artificial chemical groups.

Screening Assays

In certain embodiments, the present invention provides methods for identifying glycopeptides and/or glycopeptide epitopes reactive with disease-associated auto-antibodies using an assay comprising one or more glycopeptide panels. Preferably, such auto-antibodies bind glycopeptide epitopes through recognition of both the peptide portion and the glycan portion, but not through recognition of either the peptide or the glycan alone. The panel is contacted with a sample containing the disease-associated antibodies (e.g., sera obtained from an individual with the disease).

In a specific embodiment, the invention provides a method for identifying glycopeptides reactive with cancer-associated auto-antibodies, wherein the method comprises: (a) providing a panel comprising glycopeptides having a peptide portion and a glycan portion; (b) contacting the panel with an antibody-containing a sample from a patient with cancer; and (c) identifying glycopeptides in the panel that (i) are selectively recognized by antibodies in the sample, but not by antibodies in a control sample, and (ii) are recognized by such antibodies in the sample through recognition of both the peptide portion and the glycan portion, but not through recognition of either the peptide or the glycan alone.

Non-limiting examples of suitable antibody-containing samples for the assays of the present invention include serum, plasma, body fluids such as milk, saliva, mucosal secretions, feces, urine, cells and tissues, and any antibody preparations thereof.

Further, in order to develop the glycopeptide screening assays of the present invention, a control sample is used. By “control sample”, it is meant a sample containing pooled sera obtained from apparently disease-free or healthy individuals (i.e., individuals who do not have the disease-associated antibodies in their serum because they do not have the relevant disease), or, it is meant multiple control samples, wherein each sample is obtained from a single apparently healthy (disease-free) individual, and then data obtained in the assay for the tested control samples are compared in order to exclude any samples from control individuals who are suspected to in fact not be healthy, based on the presence of auto-antibodies not present in a statistically significant fraction of the control population.

In certain aspects, the present invention provides methods for identifying cancer-associated glycopeptide epitopes, wherein the epitope includes both part of the glycan of the glycopeptide and part of the peptide amino acid sequence. Such methods can comprise the following steps: (a) providing a panel comprising glycopeptides having a peptide portion and a glycan portion; wherein the peptide amino acid sequences of the glycopeptides are serially shifted 1-5 residues in either direction, to provide a series of overlapping peptide sequences; (b) contacting the panel with an antibody-containing sample from a patient with cancer; (c) identifying glycopeptides in the panel that (i) are selectively recognized by antibodies in the sample, but not by antibodies in a control sample, and (ii) are recognized by such antibodies in the sample through recognition of both the peptide portion and the glycan portion, but not through recognition of either the peptide or the glycan alone; and (d) mapping the minimal glycopeptide epitope based on the pattern of antibody binding to the overlapping glycopeptide sequences.

“Epitope mapping” may be carried out as follows: A glycopeptide epitope comprising e.g., a 20-mer glycopeptide with a single glycan attached to position 12 can be “mapped” in terms of peptide sequence requirement by synthesis of a panel of 20-mer glycopeptides in which each amino acid around the glycan site is modified one by one to an amino acid different from the one present in the identified glycopeptide, e.g. alanine or valine. Analysis of antibody binding to this panel of “walking” alanine or valine residues through the peptide sequence will demonstrate which residues abrogate binding, and thus provide information of the necessary peptide sequence backbone around the glycosylation site required for antibody binding.

In certain aspects of the invention, the minimum glycopeptide epitope is preferably about 2 to about 7 amino acid residues in length, but can extend to up to about 15 residues, and comprises a portion of an O-glycan modifying the glycopeptide epitope. In certain embodiments, the minimal glycopeptide epitope spans about 3 to about 4, about 2 to about 3, or about 1 to about 2 amino acid residues on each side of the O-glycan that is part of the minimal epitope identified by the methods of the present invention. In other embodiments, a greater number of amino acids may be present on one side of the O-glycan compared to the number of amino acids of the minimal epitope on the other side of the O-glycan. The minimum glycopeptide epitope can be, for example, a “minimum cancer-associated glycopeptide epitope,” which is the minimum glycopeptide epitope identified by the methods of the present invention to be specifically recognized by a cancer-associated auto-antibody.

In certain embodiments, the epitope structure of the glycopeptide epitopes identified by the present methods is determined by mass spectrometry.

In some aspects, determining whether glycopeptides and/or glycopeptide epitopes are selectively recognized by antibodies in a sample through recognition of both the peptide portion and the glycan portion, wherein the antibodies do not recognize either portion alone, may be achieved as follows: an additional panel may be provided, in which the unglycosylated forms of the glycopeptides are provided (i.e., the corresponding peptides) and antibody binding is determined. In order for a glycopeptide to be selectively recognized by an antibody in the sample, antibody binding to the corresponding peptide should be partially or preferably completely abrogated. Preferably, antibody binding to the corresponding peptide is diminished by at least about 40%, preferably at least about 50%, more preferably at least about 60%, even more preferably at least about 70%, still more preferably at least about 80%, more preferably at least about 85%, still more preferably at least about 90%, and most preferably 95% to 100% compared to antibody binding to the glycopeptide.

In other aspects of the present invention, methods of determining the presence of auto-antibodies binding O-glycopeptides, may also comprise the step of contacting the panel with a peptide inhibitor in order to exclude antibodies that recognize the non-glycosylated form of the glycopeptide. Peptide inhibitors are typically a peptide of the same amino acid sequence as the glycopeptide, however, without any glycosylations.

In other embodiments, antibodies that recognize only the hapten glycan or the glycan-conjugated to a different peptide or artificial carrier may be excluded, in order to prevent identification of epitopes that consist only of the glycan hapten. This can be achieved in several ways. For example, in certain embodiments, hapten-specific antibodies may be removed from the sample prior to analysis.

In some embodiments, hapten-specific antibodies may be removed by affinity chromatography with an appropriate resin with covalently linked carbohydrate haptens, or by inhibition with carbohydrate haptens in solution during binding assays. An example of preferable resin is GalNAc-Sepharose or other appropriate resins that would be able to bind anti-Tn hapten antibodies. Other preferable resins are GlcNAc-Sepharose, Man-Sepharose, Glc-Sepharose or Fuc-Sepharose and without limitations corresponding di- and trisaccharides in the biosynthetic pathways bound resins.

In other embodiments, hapten-specific antibodies may excluded from binding to glycopeptides in the binding assays of the present invention using a carbohydrate inhibitor. In some embodiments, the carbohydrate inhibitor comprises a normal occurring O-glycan such as sialylated or fucosylated core 2,3, or 4 O-glycan, as is typically present in normal cells. In some aspects, the carbohydrate inhibitor may be Tn, STn, T or other truncated O-glycan structures based on core 3 or 4. Also preferred are polyvalent PAA (polyacrylamide) conjugates (GlycoTech, US) of the aforementioned carbohydrates. Still in another embodiment, the carbohydrate inhibitor is a monosaccharide such as GalNAc, GlcNAc, Gal, Glc, Fuc, Man, Xyl and NeuAc. It will be apparent to the skilled artisan that other combinations of carbohydrates will have the same effect.

The above-described methods, as well as other methods that may be readily determined by a skilled artisan and used to achieve the same effect, may be used to exclude antibodies in a sample that do not specifically recognize the glycopeptide (i.e., through both the peptide and the glycan). In certain embodiments of the invention, exclusion of such antibodies from the analysis is necessary in order to identify the glycopeptides and glycopeptide epitopes that are specifically recognized by disease-associated antibodies.

In certain embodiments, a peptide inhibitor and/or an O-glycan inhibitor may be immobilized on a solid support, which is used to remove antibodies that interact with the peptide inhibitor and/or with the O-glycan carbohydrate inhibitor from a sample, prior to use of the sample according to a method of the present invention.

In some aspects, the screening assays of the present invention involve immobilized glycopeptides; however, in solution assays such as polarization, inhibition and competitive binding assays are also contemplated for use in the present invention. [See, e.g., Smith D S, Eremin S A. Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules. Anal Bioanal Chem. 2008; 391(5):1499-507.]

Non-limiting examples of screening assays involving immobilized glycopeptides contemplated for use in the present invention include antibody-binding assays, such as enzyme-linked immunosorbent assay (ELISA), multiplex bead arrays (see, Elshal et al., (2006) Methods; 38(4):317-323); BiaCore SPR analysis where binding affinities can be evaluated and microarray platforms.

In a preferred embodiment, glycopeptide libraries of the invention are immobilized on microarray slides. The glycopeptides may be printed on microarray slides, such as, e.g., Corning, Scineion or Nexterion® Slide H or Schott Nexterion® Slide H MPX 16 (Schott A G, Mainz, Germany) by JPT (Germany). Printing may be carried out using MicroGrid, ArraylT or similar according to the methods described in (Blixt O, Head S, Mondala T, Scanlan C, Huflejt M E, Alvarez R, Bryan M C, Fazio F, Calarese D, Stevens J, Razi N, Stevens D J, Skehel J J, van Die I, Burton D R, Wilson I A, Cummings R, Bovin N, Wong C H, Paulson J C. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc Natl Acad Sci USA. 2004; 101(49):17033-8)

Microarray slides of the invention may also be prepared by companies such as Schott (Louisville, Ky.), Arraylt Corp. (Sunnyvale, Calif.), or Scineon (Germany).

In certain embodiments, assays for large scale screening include, e.g., multiplex bead or array formats where many targets can be assayed simultaneously with very little consumption of antibody. For example, a small volume (50 μl) of an antibody-containing sample (e.g. serum or diluted serum) may be incubated with a solution of beads, wherein each bead is coated with a specific glycopeptide, and each bead possesses a distinguishing characteristic (e.g., size) that allows it to be differentiated from other beads in the sample. If an antibody is present in the sample that recognizes a glycopeptide on one of the beads, it will bind to the bead. Then, specific binding of antibodies to each glycopeptide in the sample may be detected using a detecting reagent, such as, e.g., biotinylated anti-Ig antibody followed by fluorescently-labeled streptavidin. The concentration of antibody in the sample that is specific for each glycopeptide on the bead may then be quantified using a bead analyzer, such as, e.g., the Luminex® 200™ System (Invitrogen).

In yet another embodiment of the invention, the detection of auto-antibodies can be limited to distinct human Ig isotypes and subclasses. Most human natural carbohydrate antibodies are of IgM isotype and it is the detection of auto-antibodies of IgG isotype and subclasses is preferred. More specifically, detection of binding of human IgG antibodies, or IgG1, 2, 3, and 4 individually, with appropriate anti-human antibodies avoid most reactivity with truncated O-glycan haptens such as Tn, STn, T core3, and other truncated structures to which IgM antibodies are found in control samples.

Methods of Diagnosing

In some aspects, the present invention provides methods for diagnosing a patient with a disease. For example, disease-associated glycopeptides and glycopeptide epitopes identified by the methods of the present invention may be used for detection of disease-associated auto-antibodies with the purpose of determining diagnosis and/or prognosis.

For example, in certain embodiments, a patient may be diagnosed as having cancer or as not having cancer, based on the presence or absence, respectively, of specific, cancer-associated glycopeptide-reactive auto-antibodies. In a specific embodiment, the method comprises contacting an antibody-containing sample from a patient with a panel comprising peptides, wherein at least a plurality of the peptides are glycopeptides. Further, each glycopeptide comprises a glycopeptide epitope that has been previously determined (i) to be selectively recognized by a subset of antibodies in sera from cancer patients, which subset recognizes neither (a) the corresponding naked peptides of said panel when not glycosylated; nor (b) the corresponding glycan when not bound to said peptide; and (ii) not to be recognized by antibodies in control sera. It is then determined if antibodies in the sample are bound to glycopeptides of said panel; and concluded either that the patient has cancer if the sample comprises antibodies that bind to at least one of the glycopeptides in the panel; or that the patient does not have cancer if the sample does not comprise antibodies that bind to at least one glycopeptide in the panel.

In certain aspects, the specific type of cancer that may be diagnosed or treated by a method of the present invention without limitation may be selected from the group consisting of breast cancer, colon cancer, ovarian cancer, cervical cancer, pancreatic cancer, prostatic cancer, liver cancer, kidney cancer, brain cancer, hematological cancers, testis cancer, head and neck cancers, and lung cancer.

In yet another embodiment, the diagnostic panel comprises glycopeptides comprising one or more and preferably at least 8 of the amino acid sequences selected from the group consisting of SEQ ID NOs. 15, 36, 49, and 82-146.

In yet another specific embodiment, the diagnostic panel comprises glycopeptides comprising one or more of the amino acid sequences selected from the group consisting of SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146). Furthermore, these glycopeptides may be O-glycosylated at one or more sites according to the methods of the present invention.

In yet another embodiment, the diagnostic panel comprises at least 8 glycopeptides, for example between about 8 and about 30 glycopeptides, such as 10, 12, 15, or 20 glycopeptides. A number of glycopeptides in excess of 30 is also within the invention. The upper limit of glycopeptides in a panel is limited by practical considerations (e.g., how many glycopeptides can fit on a substrate) or cost-benefit considerations. Preferably the glycopeptides are selected from the group consisting of SEQ ID NOs 15, 36, 49, and 82-146.

In yet other aspects, a patient may be diagnosed as having an autoimmune disease. In certain embodiments, the autoimmune disease is selected from the group consisting of coeliac disease, type I diabetes, multiple sclerosis, thyroiditis, Grave's disease, systemic lupus erythematosus, scleroderma, psoriasis, rheumatoid arthritis, alopecia greata, ankylosing spondylitis, Churg-Strauss Syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, Behcet's disease, Crohn's disease, dermatomyositis, glomerulonephritis, Guillain-Barre syndrome, inflammatory bowel disease (IBD), lupus nephritis, myasthenia gravis, myocarditis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, rheumatic fever, sarcoidosis, Sjogren's syndrome, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

Thus, in some embodiments, a diagnostic panel of the invention comprises glycopeptide epitopes determined by the methods of the present invention to be associated with an autoimmune disease. For example, in certain embodiments, a patient may be diagnosed as having an autoimmune disease or as not having an autoimmune disease, based on the presence or absence, respectively, of specific, autoimmune disease-associated glycopeptide-reactive auto-antibodies. In a specific embodiment, the method comprises contacting an antibody-containing sample from a patient with a panel comprising peptides, wherein at least a plurality of the peptides are glycopeptides. Further, each glycopeptide comprises a glycopeptide epitope that has been previously determined (i) to be selectively recognized by a subset of antibodies in sera from patients with a specific autoimmune disease, which subset recognizes neither (a) the corresponding naked peptides of said panel when not glycosylated; nor (b) the corresponding glycan when not bound to said peptide; and (ii) not to be recognized by antibodies in control sera. It is then determined if antibodies in the sample are bound to glycopeptides of said panel; and concluded either that the patient has the autoimmune disease if the sample comprises antibodies that bind to at least one of the glycopeptides in the panel; or that the patient does not have the autoimmune disease if the sample does not comprise antibodies that bind to at least one glycopeptide in the panel.

Preparation of Antibodies

Other aspects of the present invention are antibodies prepared using one or more glycopeptides identified using the methods of the present invention, methods for preparation of these antibodies, and the use of such antibodies in therapy and diagnosis.

Yet another aspect of the present invention is a method for the preparation of hybridoma cells, which secrete monoclonal antibodies specific for the glycopeptides of the invention. One such method involves immunizing a suitable mammal with a glycopeptide of the invention; fusing antibody-producing cells of the mammal with cells of a continuous cell line; the hybrid cells obtained in the fusion are cloned; and cell clones secreting the desired antibodies are selected.

Still another aspect is a monoclonal antibody selected from the group consisting of: a monoclonal antibody produced by the hybridoma cells prepared by the method described above; and a monoclonal antibody prepared by molecular display techniques, such as mRNA display, ribosome display, phage display and covalent display against a glycopeptide of the invention.

For preparation of monoclonal antibodies, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature, 1975; 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 1983; 4:72, Cote et al., Proc. Natl. Acad. Sci. U.S.A., 1983; 80:2026-2030), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO 89/12690, published 28 Dec. 1989).

According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 5,476,786 and 5,132,405 to Huston; U.S. Pat. No. 4,946,778) can be adapted to produce glycopeptide-specific single chain antibodies. Indeed, these genes can be delivered for expression in vivo to, e.g., express a glycopeptide-specific antibody. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., Science, 1989; 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for a glycopeptide or glycopeptide epitope of the invention.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

In other embodiments, alternative techniques such as mRNA display, ribosome display, phage display and covalent display may also be used. These are all display techniques where a peptide library is selected against the glycopeptide. Such techniques can e.g. be used to identify humanized or fully human antibodies.

In a preferred embodiment, the monoclonal antibody binds an O-glycoprotein containing the O-glycopeptide epitope on cancer cells but not on a non-malignant counterpart.

In another preferred embodiment, the monoclonal antibody binds preferentially to the O-glycoprotein that is aberrantly glycosylated and expressed on cancer cells.

In still another embodiment, the monoclonal antibody binds to or at least interacts directly with the O-glycopeptide.

In a preferred embodiment, the antibody prepared using a glycopeptide of the invention is humanized or fully human, such as to decrease the immunogenicity of the antibody in humans. This is typically desirable if the antibody is used as a therapeutic. However, in some situations a rapid clearance may be desired, wherefore non-humanized antibodies are also of interest as therapeutics. One such situation can, e.g., be when administering antibody conjugates where antibodies are coupled to toxins or radioisotopes. Such conjugated antibodies should either find their target rapidly or be cleared as they have a general toxic effect. Thus, one embodiment of the invention is conjugated antibodies. Non-limiting examples of antibody conjugates include radioisotopes, such as ¹³¹I, ⁹⁰Y, ¹⁷⁷Leutitium (¹⁷⁷Lu) and ⁶⁷Copper (⁶⁷Cu); toxins, such as the fungal toxin maytansanoid (DM-1); and antibiotics, such as e.g., calicheamicin [See, Ross et al. Antibody-based therapeutics: Focus on prostate cancer. Cancer and Metastasis Reviews 24: 521-537, 2005].

Compositions and Uses

In certain embodiments, the present invention provides compositions comprising one or more disease-associated glycopeptides and/or glycopeptide epitopes identified by the methods of the present invention. In certain aspects, such disease-associated glycopeptides comprise epitopes specifically recognized and bound by disease-associated antibodies.

In a specific embodiment, a composition of the invention comprises one or more of a glycoprotein or glycopeptide comprising an amino acid sequence selected from the group consisting of SEQ IDs 15, 36, 49, and 82-146. Preferably, the composition comprises one or more of a glycoprotein or glycopeptides comprising an amino acid sequence selected from the group consisting of SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146). In certain embodiments, such glycoproteins or glycopeptides are further modified at at least one amino acid residue, with an O-glycan. Preferably, the amino acid residue is serine or threonine residue.

In some aspects, the invention provides methods for eliciting an immune response in an individual, wherein the immune responses is specific for one or more glycopeptides or glycoproteins identified by the methods of the present invention.

One aspect of the invention is a method of treating cancer or an autoimmune disease comprising administering a pharmaceutical composition of the invention. In a specific embodiment, a cancer patient is treated by eliciting an anti-cancer immune response that attacks cancer cells (e.g., tumors). In any of the aspects of the invention, the immune response elicited by a pharmaceutical composition of the invention may be an adaptive T and/or B cell response (e.g., either a cytotoxic T cell response or an antibody response, or both) directed against the cancer cells, which results in reduction or elimination of the cancer cells.

“Treating” or “treatment” of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human or other mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

The benefit to an individual to be treated is either statistically significant or at least perceptible to the patient or to the physician.

Pharmaceutical Compositions and Administration

While it is possible to use a composition provided by the present invention for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Accordingly, in one aspect, the present invention provides a pharmaceutical composition or formulation comprising at least one composition of the invention, or a pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent, and/or carrier. The excipient, diluent and/or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The compositions of the invention can be formulated for administration in any convenient way for use in human or veterinary medicine.

Pharmaceutical Carrier

The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin (1990, Mack Publishing Co., Easton, Pa. 18042).

Vaccines

In certain aspects, a disease-associated glycopeptide or glycoprotein of the present invention is provided in a vaccine. Thus, in certain aspects, such a disease-associated glycopeptide or glycoprotein is provided as an “immunogen” for inducing an immune response. In some aspects, a vaccine of the present invention is useful for treating cancer by inducing an “anti-cancer immune response.” Preferably, a vaccine of the present invention in effective for inducing an adaptive immune response that selectively target cancer cells and has minimal reactivity with normal cells.

In certain embodiments, the glycopeptide epitopes identified by the methods of the present invention are targets for spontaneously-induced human auto-antibodies. Thus, it is evident that these epitopes are not generally covered by immunological tolerance. Accordingly, in certain aspects of the invention, a vaccine comprising a glycopeptide immunogen identified by the present methods does not require an adjuvant.

In other embodiments, a vaccine comprising a glycopeptide immunogen of the invention may additionally contain adjuvants to induce or enhance the desired immune response, such as, e.g., an anti-cancer immune response. Exemplary adjuvants include, but are not limited to, cholera toxin, fragments and mutants or derivatives with adjuvant properties, E. coli heat-labile enterotoxin, fragments and mutants or derivatives with adjuvant properties, oil-in-water and water-in-oil emulsions, toll-like receptor ligands such as muramyl dipeptide, E. coli LPS, oligonucleotides comprised of unmethylated DNA, poly I:C, lipoteichoic acid, peptidoglycan. Enterotoxins and their adjuvant active derivatives such as cholera toxin, heat-labile E. coli enterotoxin, pertussis toxin, shiga toxin and analogs. Other adjuvants can be used such as complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, and potentially useful human adjuvants such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine, BCG (bacille Calmette-Guerin) and Corynebacterium parvum. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p. 384). Where the vaccine is intended for use in human subjects, the adjuvant should be pharmaceutically acceptable.

Formulations

The compositions, vaccines and formulations of the present invention may comprise pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435 1712 which are herein incorporated by reference.

Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants, preserving, wetting, emulsifying, and dispersing agents. The pharmaceutical compositions may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.

Administration and Dosage

The compositions (e.g., pharmaceutical or vaccine compositions) and formulations of the present invention can be administered topically, parenterally, orally, by inhalation, as a suppository, or by other methods known in the art. The term “parenteral” includes injection (for example, intravenous, intraperitoneal, epidural, intrathecal, intramuscular, intraluminal, intratracheal or subcutaneous). The preferred routes of administration are subcutaneous and intravenous.

The compositions and formulations of the present invention may be administered to an animal, preferably a mammal, and most preferably a human.

The dosage of the compositions or formulations of the present invention will vary widely, depending upon the nature of the disease, the patient's medical history, age, body weight, sex, sensitivity, the frequency of administration, the manner and route of administration, the clearance of the agent from the host, dosage period, drugs used in combination, and the like. The initial dose may be larger, followed by smaller maintenance doses.

For any composition or formulation used in the methods of the invention, the therapeutically effective dose can be estimated initially from animal models. Dose-response curves derived from animal systems are then used to determine testing doses for the initial clinical studies in humans. In safety determinations for each composition, the dose and frequency of administration should meet or exceed those anticipated for use in the clinical studies.

Toxicity and therapeutic efficacy of the compositions, vaccines, and formulations of the invention can be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio ED₅₀/LD₅₀. Compositions that exhibit large therapeutic indices are preferred.

The data obtained from the animal studies can be used in formulating a range of doses for use in humans. The therapeutically effective doses of in humans lay preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. Ideally, a single dose of each drug should be used daily.

The compositions of the invention will typically contain an effective amount of the compositions for achieving the desired effect. As used herein, an “effective amount of a glycopeptide” is an amount that elicits the desired response upon administration, e.g., an amount that elicits an immune response in a mammal.

Administration of the compositions or formulations of the invention may be once a day, twice a day, or more often, but frequency may be decreased during a maintenance phase of the disease or disorder, e.g., once every second or third day instead of every day or twice a day. The dose and the administration frequency will depend on the clinical signs, which confirm maintenance of the remission phase, with the reduction or absence of at least one or more preferably more than one clinical signs of the acute phase known to the person skilled in the art. More generally, dose and frequency will depend in part on recession of pathological signs and clinical and subclinical symptoms of a disease condition or disorder contemplated for treatment with the present compounds.

The appropriate dose and dosage times under certain conditions can be determined by the test based on the above-described indices but may be refined and ultimately decided according to the judgment of the practitioner and each patient's circumstances (age, general condition, severity of symptoms, sex, etc.) according to standard clinical techniques.

In certain embodiments of the invention, use of an appropriate vaccine design and immunization scheme will therefore elicit immunity to a glycopeptide epitope of the immunogen and efficiency of the immunization can be monitored by immunoassays, e.g., by detecting the presence of immunogen-specific antibodies in an antibody-containing sample from an immunized patient by ELISA or other assay suitable for detecting antigen-specific antibodies.

Keeping the above description in mind, typical dosages of the glycopeptide-containing compositions of the invention are 5-50 μg glycopeptides conjugated to e.g. KLH (keyhole-Limpet Hemocyanin) given subcutaneously 3-5 times with 2-3 weeks apart. Maintenance vaccine could be extended with monthly or bi-monthly dosing for extended periods.

Kits

In some embodiments, the invention relates to a kit comprising one or more glycopeptides identified by the present methods. In certain aspects the kit comprises a panel of two or more glycopeptides identified by the present methods. In other aspects, the kit further provides instructions for use. In a specific embodiment, the kit provides a diagnostic assay for diagnosing cancer, comprising a panel of cancer-associated glycopeptides, assay buffers, and instructions for use.

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, recombinant DNA, immunology, cell biology and other related techniques within the skill of the art. See, e.g., Sambrook et al., (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al., eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacino et al., eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al., eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al., eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; Enna et al., eds. (2005) Current Protocols in Pharmacology John Wiley and Sons, Inc.: Hoboken, N.J.; Hames et al., eds. (1999) Protein Expression: A Practical Approach. Oxford University Press: Oxford; Freshney (2000) Culture of Animal Cells: A Manual of Basic Technique. 4th ed. Wiley-Liss; among others. The Current Protocols listed above are updated several times every year.

EXAMPLES

The present invention is described further below in working examples which are intended to further describe the invention without limiting the scope therein.

Example 1 Development of a Cancer-Associated O-Glycopeptide Library for Microarray Display

GalNAc-Ser/Thr-Fmoc amino acids were synthesized by Sussex Inc (Canada) and 20-mer peptides and corresponding glycopeptides with one GalNAc O-glycan synthesized and printed on epoxy activated glass slides by JPT Peptide Technologies (Berlin, Germany). A total of 48 paired peptides and 48 GalNAc-glycopeptides were synthesized and printed (Table II).

TABLE II Synthetic 96 paired glycopeptide 20-mer library derived  from confirmed O-glycoproteins* SEQ ID Peptide SEQ ID Peptide NO: No. NO: No.  4  1 TSAPDTRPAPGSTAPPAHGV 29 49 CDKDKTSTVAP T IHTTVPSP  4  2 TSAPDTRPAPG S TAPPAHGV 30 50 DKTSTVAPTIHTTVPSPTTT  5  3 SAPDTRPAPGSTAPPAHGVT 30 51 DKTSTVAPTIH T TVPSPTTT  5  4 SAPDTRPAPGS T APPAHGVT 31 52 KTSTVAPTIHTTVPSPTTTP  6  5 PGSTAPPAHGVTSAPDTRPA 31 53 KTSTVAPTIHT T VPSPTTTP  6  6 PGSTAPPAHGV T SAPDTRPA 32 54 TVAPTIHTTVPSPTTTPTPK  7  7 GSTAPPAHGVTSAPDTRPAP 32 55 TVAPTIHTTVP S PTTTPTPK  7  8 GSTAPPAHGVT S APDTRPAP 33 56 APTIHTTVPSPTTTPTPKEK  8  9 PPAHGVTSAPDTRPAPGSTA 33 57 APTIHTTVPSP T TTPTPKEK  8 10 PPAHGVTSAPD T RPAPGSTA 34 58 PTIHTTVPSPTTTPTPKEKP  9 11 FYLAMPFATPMEAELARRSL 34 59 PTIHTTVPSPT T TPTPKEKP 10 12 KFSEFWDLDPEVRPTSAVAA 35 60 TIHTTVPSPTTTPTPKEKPE 11 13 PLVEQGRVRAATVGSLAGQP 35 61 TIHTTVPSPTT T PTPKEKPE 11 14 PLVEQGRVRAA T VGSLAGQP 36 62 TPTPKEKPEAGTYSVNNGND 12 15 LWSLCWSLAIATPLPPTSAH 36 63 TPTPKEKPEAG T YSVNNGND 12 16 LWSLCWSLAIA T PLPPTSAH 37 64 ACLAVSAGPVPTPPDNIQVQ 13 17 LEACVIQGVGVTETPLMKED 37 65 ACLAVSAGPVP T PPDNIQVQ 13 18 LEACVIQGVGV T ETPLMKED 38 66 RRAVLPQEEEGSGGGQLVTE 14 19 KKWVQDSMKYLDQKSPTPKP 38 67 RRAVLPQEEEG S GGGQLVTE 15 20 SHHSDESDELVTDFPTDLPA 39 68 LNAVNNSLTPQSTKVPSLFE 15 21 SHHSDESDELV T DFPTDLPA 39 69 LNAVNNSLTPQ S TKVPSLFE 16 22 DESDELVTDFPTDLPATEVF 40 70 NAVNNSLTPQSTKVPSLFEF 16 23 DESDELVTDFP T DLPATEVF 40 71 NAVNNSLTPQS T KVPSLFEF 17 24 FPTDFPTDLPATEVFTPVVP 41 72 TFVLSALQPSPTHSSSNTQR 17 25 FPTDFPTDLPA T EVFTPVVP 41 73 TFVLSALQPSP T HSSSNTQR 18 26 FPTDLPATEVFTPVVPTVDT 42 74 RQGWALRPVLPTQSAHDPPA 18 27 FPTDLPATEVF T PVVPTVDT 42 75 RQGWALRPVLP T QSAHDPPA 19 28 PATEVFTPVVPTVDTYDGRG 43 76 QKKAKNLDAITTPDPTTNAS 19 29 PATEVFTPVVP T VDTYDGRG 43 77 QKKAKNLDAIT T PDPTTNAS 20 30 PGAQGLPGVGLTPSAAQTAR 44 78 FLSLSQGQESQTELPNPRIS 20 31 PGAQGLPGVGL T PSAAQTAR 44 79 FLSLSQGQESQ T ELPNPRIS 21 32 RGETRCEQDRPSPTTAPPAP 45 80 LSLALVTNSAPTSSSTKKTQ 21 33 RGETRCEQDRP S PTTAPPAP 45 81 LSLALVTNSAP T SSSTKKTQ 22 34 ETRCEQDRPSPTTAPPAPPS 46 82 LISPLAQAVRSSSRTPSDKP 22 35 ETRCEQDRPSP T TAPPAPPS 46 83 LISPLAQAVRS S SRTPSDKP 23 36 TRCEQDRPSPTTAPPAPPSP 47 84 DDENTAQFVHVSESFPHPGF 23 37 TRCEQDRPSPT T APPAPPSP 47 85 DDENTAQFVHV S ESFPHPGF 24 38 PSPTTAPPAPPSPSPSPVPK 48 86 SESFPHPGFNMSLLENHTRQ 24 39 PSPTTAPPAPP S PSPSPVPK 48 87 SESFPHPGFNM S LLENHTRQ 25 40 PTTAPPAPPSPSPSPVPKSP 49 88 SGWGSIEPENFSFPDDLQCV 25 41 PTTAPPAPPSP S PSPVPKSP 49 89 SGWGSIEPENF S FPDDLQCV 26 42 TAPPAPPSPSPSPVPKSPSV 50 90 RIQRGPGRAFVTIGKIGNMR 26 43 TAPPAPPSPSP S PVPKSPSV 50 91 RIQRGPGRAFV T IGKIGNMR 27 44 STNEFLCDKDKTSTVAPTIH 51 92 EMSRHSLEQKPTDAPPKVLT 27 45 STNEFLCDKDK T STVAPTIH 51 93 EMSRHSLEQKP T DAPPKVLT 28 46 NEFLCDKDKTSTVAPTIHTT 52 94 CSESLELEDPSSGLGVTKQD 28 47 NEFLCDKDKTS T VAPTIHTT 52 95 CSESLELEDPS S GLGVTKQD 29 48 CDKDKTSTVAPTIHTTVPSP 53 96 LLEFYLAMPFA T PMEAELAR *Indicates attachment site of GalNAc residues to Ser/Thr (bold, underlined).

Glycopeptides were synthesized with and without N-terminal linker for printing on epoxy or NHS-activated glass slides, respectively. Peptides were designed based on known O-glycoproteins using the algorithm NetOGlyc for selection of O-glycosylation sites (Julenius et al. 2005), which in most cases coincides with experimentally determined O-glycosylation found on isolated proteins, as these glycoproteins have served as a training set for the algorithm. Glycopeptides were designed with a single GalNAc at position 12 to allow optimal exposure of peptide sequence flanking the O-glycan taking into consideration that the glycopeptides will be covalently linked primarily through the N-terminal amino acid. Glycopeptides were synthesized at 100 nmol scale using step-wise blocking by acetylation and printing directly with theoretical excess of 50 fold onto slides.

In one group, the paired peptide and single GalNAc glycopeptide library was further expanded by on-slide glycosylation with one or more recombinant glycosyltransferases to enhance the number of GalNAc O-glycosylation sites using one or more recombinant polypeptide GalNAc-transferases (e.g., GalNAc-T2, -T3 and -T4) (see, U.S. Pat. Nos. 5,876,716 and 6,465,220) (Bennett et al. 1998; Bennett et al. 1996; White et al. 1995) (see also, FIG. 1). Further expansion was achieved by use of a recombinant sialyltransferase, ST6GalNAc-II, to produce STn glycoforms (structure no. 2, Table I) of the GalNAc glycopeptides. T glycoforms were produced by a recombinant Drosophila core1 β3galactosyltransferase, truncated core3 glycoforms (structure no. 6, Table I) were produced with a recombinant human β3GlcNAc-transferase (Iwai et al. 2002), and non-capped type1-core3 glycoforms (structure no. 9, Table I) were produced using β3Gal-T5 (see, U.S. Pat. No. 7,332,279).

Recombinant enzymes were expressed in insect cells using the baculo-virus system and used after semi-purification by Ni-chromatography (when HIS-tagged) or by ion exchange chromatographies. Glycosylation was monitored by staining of slides with lectins (HPA, VVA) (Sigma) and monoclonal antibodies to Tn (HBTn1, HBSTn1, HBT1) (Dako, Denmark). On-slide glycosylation was performed as follows: slides were quenched for 1 hr in 50 mM ethanolamine in 100 mM sodium borate pH 8, washed extensively in de-ionized water, and spun dry. Slides were blocked for 1 h with 1% BSA in PBS, pH 7.4, and in some cases with NP40 (1%) to reduce background. Slides were washed in PBS/.05% Tween and enzyme reaction mixtures with BSA and detergent were applied and incubated 1-2 hrs at 37° C. Reaction mixtures for polypeptide GalNAc-transferases included MES buffer 125 mM, pH 7.4, 1% NP40, 1% BSA, 250 μM UDP-GalNAc, 2 mM MnCl₂ and 20 μg/mL enzyme. Reaction mixtures for galactosyltransferases included UDP-Gal, sialyltransferases CMP-NeuAc, GlcNAc-transferases UDP-GlcNAc. Following on-slide glycosylation slides were washed with PBS/Tween and processed with antibodies and lectins as described below.

It will be clear to one of ordinary skill in the art that on-slide glycosylation serves as a method to screen for additional glycoforms of peptides which may be recognized by auto-antibodies present in patients. Once a particular glycoform of a given peptide is found to react with an autoantibody from a patient, the glycopeptide can be resynthesized in solution using the same enzymes used for on-slide glycosylation and the glycan structure and sites of attachment in the peptide confirmed by mass spectrometry (Tarp et al. 2007). The validated glycopeptide can then be incorporated into diagnostic panels as described throughout the specification and in Example 4.

FIG. 1 illustrates an example of on-slide glycosylation with the polypeptide GalNAc-transferase, GalNAc-T3, to glycosylate peptides and GalNAc glycopeptides having additional unsubstituted Ser/Thr residues to enhance the number of O-glycans. A library of 96 paired GalNAc-glycopeptides/peptides (20-mers with and without a single GalNAc residue at position 12), as designated in Table II, supra, were printed in triplicates (horizontal) on Scineon 16-well slides by JPT (Germany). In each pair in Table II, the glycopeptide of the pair contains a bold, underlined amino acid residue, indicating the site of attachment of GalNAc. Peptide pairs by peptide number are as follows: 1/2, 3/4, 5/6 . . . etc. The slide was reacted with the anti-Tn lectin HPA (1 μg/ml) without (FIG. 1A) and with (FIG. 1B) prior treatment with recombinant polypeptide GalNAc-T3 enzyme for on-slide GalNAc-glycosylation of available unglycosylated sites on peptides and glycopeptides.

The HPA lectin did not react with all GalNAc-glycopeptides (e.g., glycopeptide nos. 1-5 and 25-29), which is partly due to failure in synthesis and/or printing at these positions, as well as some restrictions of specificity of the GalNAc-binding lectin. The GalNAc-glycopeptides 1-5 did react with the HPA lectin in other experiments, and it was therefore concluded that there was a print failure. Conversion of peptides to GalNAc-glycopeptides by on-slide reaction with a polypeptide GalNAc-transferase is also expected to be dependent on the substrate specificity of the enzyme used, and, e.g., use of GalNAc-T2 rather than T3 may give a partly different labeling pattern.

Regardless, it is evident that most of the GalNAc-glycopeptides were labeled with HPA (FIG. 1A) and that most of the corresponding paired peptides were labeled only after GalNAc-T3 on-slide glycosylation (FIG. 1B). While HPA may not be expected to react with all GalNAc-glycopeptides, this lectin and other Tn reactive lectins such as HAA, VVA and DBA, as well as monoclonal anti-Tn antibodies (Dako) provide excellent controls for determining the quality of peptide synthesis and printing.

Example 2 High Through-Put Screening of Glycopeptide Microarrays with Human Serum for Identification of Glycopeptides Recognized by Cancer-Associated Auto-Antibodies

Sera Origin and Handling:

A panel of human sera was obtained from CHTN (Cooperative Human Tissue Network) and Asterand Inc., under the guidelines of approved agreements by the providers. Sera obtained were from control individuals (“normal”) (n=31) and from newly diagnosed cancer patients (n=147) with pancreatic, breast, colon, lung, prostate or ovary primary cancers. Briefly, for cancer sera, all blood samples were obtained on or near date of diagnosis, and serum was processed immediately, flash frozen and stored at −70° C. until shipment. Serum samples received from the providers as frozen aliquots generally of 1-2 mL were brought to room temperature, vortexed and distributed into 20-100 μL aliquots in closeable Eppendorf tubes (vWR) (pre-labeled), and immediately frozen and stored at −70° C. until use.

Glycopeptide Microarray Method:

Glycopeptides (20-mers with and without a single GalNAc residue at position 12) and control structures (corresponding unglycosylated peptides) were printed on Corning (Corning, N.Y.), Scienion (Germany) or Schott Nexterion® Slide H or Schott Nexterion® Slide H MPX 16 (Schott A G, Mainz, Germany) by JPT (Germany). Triplicates or quadruplicates of all compounds were printed at optimal concentrations (1-50 μM) or 50× excess relative to scale of synthesis for spot-synthesized glycopeptides printed without purification. After printing, slides were incubated for 1 hour (h) in a humidified hybridization chamber with 70-100% relative humidity and stored until use at 4° C. Unspotted slide areas were blocked for 1 h with 25 mM ethanolamine in 100 mM sodium borate pH 8.5. If an enzyme step was needed either to increase sites of GaLNac attachments in peptides or extend O-glycans to T, STn, core 3 or other structures, addition of the enzyme reaction mixture in 25-35 μL was made in appropriate wells and the slide incubated at 37° C. for 1 h, after which it was washed in PBS/.05% Tween and then PBS, and then spun dry.

Human sera (usually diluted 1:25), monoclonal antibodies, lectins and the like were added in 25 μL and the slide left at room temperature for 2 h in a moist, humid chamber after which it was washed as above and spun dry (if no superstructure). Secondary antibodies were added at appropriate concentrations (for human sera, usually 1:1500 for anti-human IgG with a Cy3 chromophore). In some cases, for some controls, lectin-Cy3 was used directly as final step. The final step was washing as above, with a brief de-ionized water wash and the slide spun dry for scanning Analysis was made on a GenePix 4200AL Scanner at PMT 400 and power mode 10-50. Data were analyzed and plotted using Microsoft Excel.

Identification of Cancer-Associated Auto-Antibodies to GalNAc-Glycopeptide Epitopes:

Glycopeptides (20-mers with and without a single GalNAc residue at position 12) were printed in triplicates (horizontal) on Scineon 16-well slides by JPT (Germany) and bound human IgG antibodies detected by a labeled secondary anti-human IgG antibody. FIG. 2 illustrates examples of reactivities of serum (1:25 dilution) from a newly diagnosed prostate cancer patient (#762 in Panel A) and a normal control serum from a healthy individual (#174 in Panel B) on the library of 96 paired peptides/GalNAc-glycopeptides as designated in Table II. Candidate cancer-associated IgG antibodies identified in the cancer serum directed to GalNAc glycopeptide epitopes are indicated by open circles labeled 20/21, 62/63, and 86/87 for the paired peptide and GalNAc glycopeptides, respectively. The analysis demonstrates that serum of cancer patient contain IgG antibodies specifically reacting with epitopes found on GalNAc glycopeptides 21, 63, and 87, and since these antibodies do not react with the corresponding unglycosylated peptides 20, 62, and 86, it may be concluded that the epitopes are comprised of a GalNAc-peptide epitope including both the O-glycan part as well as part of the peptide sequence.

Human IgG antibodies from several cancers were shown to bind selectively to several GalNAc-glycopeptides and not the corresponding peptide, and such antibodies were not detected in healthy individuals. An example of this is shown in FIG. 2, where a prostate cancer serum (prca#762) labels three GalNAc-glycopeptides (#21, 63, and 87), and not the corresponding peptide (#20, 62, and 86). These three candidates were also identified in other cancer sera but not in the controls as shown in FIGS. 3-5, and described, infra.

FIG. 3 illustrates results of screening 147 cancer sera and 31 control sera (normal sera) on the glycopeptide pair #20/21. Sera obtained from newly diagnosed cancer patients with lung, colon, ovary, prostate, pancreas or breast tumors as well as controls (as indicated) were reacted with the 96-peptide array on Scineon 16-well slides (1:25 dilution) followed by cy3 labeled anti-human IgG (diluted to 1:1500). Arrays were analyzed on GenePix 4200 scanner at 400 pmt with 50 power and relative intensities graphed.

FIG. 4 illustrates results of screening 147 cancer sera and 31 normal sera on the glycopeptide pair #62/63. FIG. 5 illustrates results of screening 147 cancer sera and 31 normal sera on the glycopeptide pair #86/87.

Peptides 20/21, each having the sequence identified as SEQ ID NO: 15, are derived from SPP1 (Ensg00000118785) GenBank Accession No. NP_001035147 (SEQ ID NO: 61), peptides 62/63, each having the sequence identified as SEQ ID NO: 36 are derived from LAMP2 (Ensg00000005893) (ENST00000200639) protein (ENSP00000200639) GenBank Accession No. NP_002285 (SEQ ID NO: 64), and peptides 86/87, each having the sequence identified as SEQ ID NO: 49, are derived from KLK1 (gene: Ensg00000167748) (transcript: ENST00000301420) (protein: ENSP00000301420) (GenBank Accession No. NP_002248) (SEQ ID NO: 66). These proteins have broad expression patterns in tissues and hence may induce antibodies in many cancers.

The presented method using a limited glycopeptide library identified three candidate targets for human disease-associated auto-antibodies. These auto-antibody targets were defined by the discriminating factor that cancer-associated IgG antibodies reacted selectively with a glycopeptide and not the corresponding peptide or other glycopeptides with the same O-glycan or the O-glycan presented as a hapten on an artificial carrier.

Example 3 Generation of Antibodies to Identified Auto-Antibody Targets for Diagnostic Use

Monoclonal or polyclonal antibodies may be generated to the identified glycopeptide antigens by known methods (Takeuchi et al. 2002; Hanisch et al. 1995; Reis et al. 1998; Sorensen et al. 2006). Briefly, the following procedure serves as an example for generation of antibodies with the desired glycopeptides specificity but other procedures leading to the same result will be known to the skilled in the art.

Immunization protocol: Glycopeptides are coupled to keyhole limpet hemocyanin (KLH) (Pierce, Rockford, Ill.) using glutaraldehyde. Efficiency of conjugation is assessed by analyzing the reaction by size exclusion chromatography on a PD-10 column, where the conjugate/glycopeptides ratio can be determined by ELISA using appropriate reagents such as antibodies and lectins detecting the glycopeptides. Essentially all reactivity with a Tn reactive lectin (HPA) is found with the excluded fraction and insignificant reactivity in the included fractions expected to contain peptides. Titration analysis of the KLH conjugate with the corresponding glycopeptide in ELISA indicated conjugation ratio KLH to glycopeptide of approximately 1:200. Female Balb/c (Jackson Labs) wild type mice are injected subcutaneously with 10 or 15 μg of glycopeptide-KLH in a total volume of 200 μl (1:1 mix with Freunds adjuvant, Sigma). Mice received four immunizations 2-4 wks apart, and blood samples are obtained by tail or eye bleeding 1 wk following the third and fourth immunization.

Hybridoma Production:

Mouse hybridomas are produced by fusion of splenocytes to NS-1 followed by selection in HAT/HT (Hypoxanthine, Aminopterin, Thymidine). Hybridomas are selected by initial screening by ELISA with GalNAc-glycopeptides and corresponding peptides as well as irrelevant control compounds. Further characterization is done on glycopeptides microarrays as well as on a panel of human cancer cell lines expressing the corresponding aberrant glycoprotein.

Enzyme-linked immunosorbent assays (ELISA) are performed using 96-well MaxiSorp™ plates (Nunc). Plates are coated overnight at 4° C. with 1 μg/ml of glycopeptides in bicarbonate-carbonate buffer (pH 9.5), blocked with 5% BSA in PBS, followed by incubation with sera (diluted in PBS) or monoclonal antibodies for 2 hours (hrs) at room temperature. Bound antibodies are detected with peroxidase-conjugated rabbit anti-mouse immunoglobulins (Dako, Denmark) or isotype specific antibodies peroxidase-conjugated goat anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology Associates, USA). Plates are developed with o-phenylenediamine tablets (Dako, Denmark) and read at 492 nm. Control antibodies included anti-carbohydrate antibodies HBTn-1 (Tn) and HBSTn-1 (STn) (Dako) and lectins HPA, VVA and HAA (Sigma). Control sera included mice immunized with irrelevant peptides linked to KLH. Human cancer cell lines from different cancers are all obtained from ATCC and cultured as recommended by ATCC.

Immunocytochemical staining of cells are performed as follows: cells are harvested by trypsination, washed and plated onto multi-well glass slides and fixed for 10 minutes (min) in ice cold acetone or in methanol acetone. Fixed cells are incubated overnight at 4° C. with mouse sera (1:200/1:400/1:800) or hybridoma antibodies, followed by incubation for 45 min at room temperature with FITC-conjugated rabbit anti-mouse immunoglobulins (Dako, Denmark). Slides are mounted in glycerol containing p-phenylenediamine and examined in a fluorescence microscope.

For further determination of expression of glycopeptides epitope in human cancers immunohistochemistry of fixed and frozen tissue samples are performed. Frozen sections are fixed for 10 min in cold methanol/acetone (50:50). Formalin fixed, paraffin wax embedded tissues of different carcinoma and healthy tissues are obtained from Origine (US) and stained by immunofluorescence or peroxidase techniques. Paraffin sections are dewaxed, rehydrated, and treated with 0.5% H₂O₂ in methanol for 30 min. Section are rinsed in TBS and incubated for 20 min with rabbit nonimmune serum. Sections are rinsed and incubated overnight at 4° C. with primary antibody. Sections are rinsed and incubated with biotin-labeled rabbit anti-mouse serum (Dako, Denmark) diluted 1:200 in TBS for 30 min, rinsed with TBS, and incubated for 1 h with avidin-biotin-peroxidase complex (Dako, Denmark). Sections are rinsed with TBS and developed with 0.05% 3,3′-diaminobenzidine tetrahydrochloride freshly prepared in 0.05 M TBS containing 0.1% H₂O₂. Sections are stained with hematoxylin, dehydrated and mounted.

Example 4 Broad Discovery of O-Glycopeptide Epitopes

A larger library of GalNAc O-glycopeptides designed with GalNAc linked to serine or threonine at position 12 in 20-mer peptides was produced as described in Example 1. Peptides were selected among cell membrane and secreted human proteins that contain serine and threonine residues that are predicted to be O-glycosylated by the NetOGlyc algorithm (Julenius et al. 2005). A total of 960 GalNAc glycopeptides were synthesized at 0.5 mg scale (Sigma) and printed on microarray hydrogel slides (Schott Nexterion) as described in Example 1. Slides were reacted with human cancer sera (ovarian, breast, colon, lung, pancreas, prostate, 235 total sera) and control sera (145 healthy, 20 inflammatory, 20 benign tumor). Glycopeptides preferentially reactive with human IgG antibodies from cancer patients compared to controls were selected for resynthesis at 5 mg scale with amidated C-terminus (New England Peptide (Gardner, Mass.) (Table III). Resynthesized glycopeptides were reprinted on microarray slides at 50 μM and reacted with human cancer sera (Total 286 sera: 32 ovarian cancer (O), 38 breast cancer (B), 54 colon cancer (C), 17 lung cancer (L), and 145 age and sex matched control normal sera as described in Example 1. Table III lists glycopeptides exclusively or preferentially reactive with human IgG from cancer patients compared to controls and examples of reactivities are shown in FIG. 6.

TABLE III Glycopeptides reactive with IgG antibodies from human cancer sera Peptide  Sequence number ID Protein Sequence 166  82 Oncostatin H2N-TKAGRGASQPPTPTPASDAF-amide 113  83 gp95 H2N-EEEPEETAEDTTEDTEQDED-amide 218  84 MUC13 H2N-TASTTANTPFPTATSPAPPI-amide 225  85 MUC13 H2N-PAPPIISTHSSSTIPTPAPP-amide 275a  86 Ceruplasm H2N-LAKMYYSAVEPTKDIFTGLI-amide 284  87 CD46 H2N-PSSTKPPALSHSVSTSSTTK-amide 308  88 EBAG9 H2N-LEPDYFKDMTPTIRKTQKIV-amide 318a  89 MUC17 H2N-STMPVVSSEASTHSTTPVDT-amide 324  90 MUC17 H2N-STHSTTPVDTSTPVTTSTEA-amide 370  91 IL6-R H2N-IPPEDTASTRSSFTVQDLKP-amide 382  92 R-PTP-alpha H2N-EAKTSNPTSSLTSLSVAPTF-amide 389  93 R-PTP-alpha H2N-ARTEPWEGNSSTAATTPETF-amide 443  94 ODAM H2N-VDPLQLQTPPQTQPGPSHVM-amide 450  95 ODAM H2N-SPKPSTTNVFTSAVDQTITP-amide 456  96 IGFB-3 H2N-PAPPAPGNASESEEDRSAGS-amide 458  97 IGFB-3 H2N-ASESEEDRSAGSVESPSVSS-amide 465  98 MUC15 H2N-ANLNSDKENITTSNLKASHS-amide 484  99 MUC15 H2N-LTTNSDSFTGFTPYQEKTTL-amide 485 100 MUC15 H2N-SFTGFTPYQEKTTLQPTLKF-amide 485a 101 MUC15 H2N-SFTGFTPYQEKTTLQPTLKF-amide 505 102 TPBG Trophob H2N-SPTSSASSFSSSAPFLASAV-amide 522 103 IgA1 hinge H2N-TVPCPVPSTPPTPSPSTPPT-amide 524 104 IgA1 hinge H2N-VPSTPPTPSPSTPPTPSPSC-amide 544 105 R-PTP-N H2N-SEPPKAARPPVTPVLLEKKS-amide 546 106 R-PTP-N H2N-GQSQPTVAGQPSARPAAEEY-amide 558 107 CMRF35 H2N-TPASITAAKTSTITTAFPPV-amide 569 108 TNF-RSF1B H2N-GNASMDAVCTSTSPTRSMAP-amide 585 109 CGB2 H2N-TDCGGPKDHPLTCDDPRFQA-amide 587 110 CGB2 H2N-AQASSSSKAPPPSLPSPSRLP-amide 592 111 Acrosomal SP H2N-PLSELESGEQPSDEQPSGEH-amide 601 112 Cadherin 1 H2N-PQRSSTAILQVSVTDTNDNH-amide (CD) 605 113 CD Ovomorolin H2N-GALPGTSVMEVTATDADDDV-amide 612 114 CD Mucin like H2N-EQEPPSTDVPPSPEAGGTTG-amide 676 115 Inhibin alpha H2N-RPEATPFLVAHTRTRPPSGG-amide 690 116 IGF-BP-6 H2N-PGTSTTPSQPNSAGVQDTEM-amide 739 117 LTBP1 H2N-EVAPEASTSSASQVIAPTQV-amide 752 118 CD 248 H2N-QPPDFALAYRPSFPEDREPQ-amide Endosialin  755 119 CD 248 H2N-LSVTRPVVVSATHPTLPSAH-amide Endosialin  757 120 CD 248 H2N-PSAHQPPVIPATHPALSRDH-amide Endosialin  765 121 CD 248 H2N-APDALVLRTQATQLPIIPTA-amide Endosialin  823 122 ICAM-1 H2N-GALFPGPGNAQTSVSPSKVI-amide 827 123 ICAM-1 H2N-HLALGDQRLNPTVTYGNDSF-amide 827a 124 ICAM-1 H2N-HLALGDQRLNPTVTYGNDSF-amide 848 125 MUC1 H2N-PATEPASGSAATWGQDVTSV-amide 852 126 MUC1 H2N-VPVTRPALGSTTPPAHDVTS-amide 859 127 MUC1 H2N-NVTSASGSASGSASTLVHNG-amide 859a 128 MUC1 H2N-NVTSASGSASGSASTLVHNG-amide 863 129 MUC1 H2N-GTSARATTTPASKSTPFSIP-amide 863a 130 MUC1 H2N-GTSARATTTPASKSTPFSIP-amide 870 131 MUC1 H2N-SDTPTTLASHSTKTDASSTH-amide 873 132 MUC1 H2N-TKTDASSTHHSTVPPLTSSN-amide 883 133 MUC1 H2N-TDYYQELQRDISEMFLQIYK-amide 889 134 MUC1 H2N-HDVETQFNQYKTEAASRYNL-amide 893 135 MUC1 H2N-ASRYNLTISDVSVSDVPFPF-amide 894 136 MUC1 H2N-RYNLTISDVSVSDVPFPFSA-amide 895 137 MUC1 H2N-DVSVSDVPFPFSAQSGAGVP-amide 914 138 MUC4 H2N-TAGRPTGQSSPTSPSASPQE-amide 931 139 MUC4 H2N-SLASQATDIFSTVPPTPPSI-amide 934 140 MUC4 H2N-FSTVPPTPPSITSTGLTSPQ-amide 936 141 MUC4 H2N-PTPPSITSTGLTSPQTETHT-amide 941 142 MUC4 H2N-LTSPQTETHTLSPSGSGKTF-amide 977 143 MUC4 H2N-TDTSSASTGHATPLPVTSLS-amide 983 144 MUC4 H2N-HATPLAVSSATSASTVSSDS-amide 852-C3 145 MUC1 H2N- VPVTRPALGSTT[GlcNAc]PPAHDVTS- amide 931-C3 146 MUC4 H2N- SLASQATDIFST[GlcNAc]VPPTPPSI- amide *Bold S or T indicates attachment site of GalNAc residues to Ser/Thr; Bold [GlcNAc] indicates an addition of glucosamine attached to GalNAc (in bold). Bold, underlined name indicates peptide assayed in graphs in FIG. 6.

Since IgG antibodies reactive with glycopeptides epitopes recognize both the peptide sequence in close proximity to the O-glycan (i.e., one to five or even eighth residues N- and C-terminal of the O-glycan) as well as the O-glycan structure, it is clear that different glycoforms of one glycopeptide may be recognized by different IgG antibodies and these antibodies may be found in different cancer patients. In this Example, GalNAc-glycopeptides from MUC1 and MUC4 were also produced as the core 3 GlcNAcβ1-3GalNAcα1-O-Ser/Thr glycoform with a recombinant core 3 synthase as described in Example 1. Two core 3 glycopeptides (#852-C3 and #931-C3) were identified as exclusively recognized by IgG antibodies in sera from cancer patients.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all values are approximate, and are provided for description.

Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

LITERATURE CITED

-   Anderson K S, Labaer J (2005) The sentinel within: exploiting the     immune system for cancer biomarkers. J Proteome Res 4:1123-1133. -   Anderson K S, Ramachandran N, Wong J, Raphael J V, Hainsworth E,     Demirkan G, Cramer D, Aronzon D, Hodi F S, Harris L, Logvinenko T,     Labaer J (2008) Application of protein microarrays for multiplexed     detection of antibodies to tumor antigens in breast cancer. J     Proteome Res 7:1490-1499. -   Anderton S M (2004) Post-translational modifications of self     antigens: implications for autoimmunity. Curr Opin Immunol     16:753-758. -   Backlund J, Treschow A, Bockermann R, Holm B, Holm L,     Issazadeh-Navikas S, Kihlberg J, Holmdahl R (2002) Glycosylation of     type II collagen is of major importance for T cell tolerance and     pathology in collagen-induced arthritis. Eur J Immunol 32:3776-3784. -   Bennett E P, Hassan H, Clausen H (1996) cDNA cloning and expression     of a novel human UDP-N-acetyl-alpha-D-galactosamine—Polypeptide     N-acetylgalactosaminyltransferase, GalNAc-T3. Journal of Biological     Chemistry 271:17006-17012. -   Bennett E P, Hassan H, Mandel U, Mirgorodskaya E, Roepstorff P,     Burchell J, Taylor-Papadimitriou J, Hollingsworth M A, Merkx G, van     Kessel A G, Eiberg H, Steffensen R, Clausen H (1998) Cloning of a     human UDP-N-acetyl-alpha-D-Galactosamine:polypeptide     N-acetylgalactosaminyltransferase that complements other     GalNAc-transferases in complete O-glycosylation of the MUC1 tandem     repeat. J Biol Chem 273:30472-30481. -   Brandlein S, Eck M, Strobel P, Wozniak E, Muller-Hermelink H K,     Hensel F, Vollmers H P (2004a) PAM-1, a natural human IgM antibody     as new tool for detection of breast and prostate precursors. Hum     Antibodies 13:97-104. -   Brandlein S, Pohle T, Vollmers C, Wozniak E, Ruoff N,     Muller-Hermelink H K, Vollmers H P (2004b) CFR-1 receptor as target     for tumor-specific apoptosis induced by the natural human monoclonal     antibody PAM-1. Oncol Rep 11:777-784. -   Chapman C, Murray A, Chakrabarti J, Thorpe A, Woolston C, Sahin U,     Barnes A, Robertson J (2007) Autoantibodies in breast cancer: their     use as an aid to early diagnosis. Ann Oncol 18:868-873. -   Clark R A, Fuhlbrigge R C, Springer T A (1998) L-Selectin ligands     that are O-glycoprotease resistant and distinct from MECA-79 antigen     are sufficient for tethering and rolling of lymphocytes on human     high endothelial venules. J Cell Biol 140:721-731. -   Danielczyk A, Stahn R, Faulstich D, Loffler A, Marten A, Karsten U,     Goletz S (2006) PankoMab: a potent new generation anti-tumour MUC1     antibody. Cancer Immunol Immunother 55:1337-1347. -   Dian D, Janni W, Kuhn C, Mayr D, Karsten U, Mylonas I, Friese K,     Jeschke U (2009) Evaluation of a novel anti-mucin 1 (MUC1) antibody     (PankoMab) as a potential diagnostic tool in human ductal breast     cancer; comparison with two established antibodies. Onkologie     32:238-244. -   Doyle H A, Mamula M J (2001) Post-translational protein     modifications in antigen recognition and autoimmunity. Trends     Immunol 22:443-449. -   Doyle H A, Mamula M J (2005) Posttranslational modifications of     self-antigens Ann N Y Acad Sci 1050:1-9. -   Gahring L, Carlson N G, Meyer E L, Rogers S W (2001) Granzyme B     proteolysis of a neuronal glutamate receptor generates an     autoantigen and is modulated by glycosylation. J Immunol     166:1433-1438. -   Hanisch F G, Stadie T, Bosslet K (1995) Monoclonal-Antibody Bw835     Defines A Site-Specific Thomsen-Friedenreich Disaccharide Linked to     Threonine Within the Vtsa Motif of Muc1 Tandem Repeats. Cancer     Research 55:4036-4040 -   Hassan H, Bennett E P, Mandel U, Hollingsworth M A, Clausen H (2000)     Carbohydrates in Chemistry and Biology—a Comprehension Handbook.     Wiley-VCH, pp. 273-292. -   Hellstrom I, Friedman E, Verch T, Yang Y, Korach J, Jaffar J,     Swisher E, Zhang B, Ben Baruch G, Tan M C, Goedegebuure P, Hellstrom     K E (2008) Anti-mesothelin antibodies and circulating mesothelin     relate to the clinical state in ovarian cancer patients. Cancer     Epidemiol Biomarkers Prev 17:1520-1526. -   Iwai T, Inaba N, Naundorf A, Zhang Y, Gotoh M, Iwasaki H, Kudo T,     Togayachi A, Ishizuka Y, Nakanishi H, Narimatsu H (2002) Molecular     cloning and characterization of a novel UDP-GlcNAc:GalNAc-peptide     beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T6), an enzyme     synthesizing the core 3 structure of O-glycans. J Biol Chem     277:12802-12809. -   Julenius K, Molgaard A, Gupta R, Brunak S (2005) Prediction,     conservation analysis, and structural characterization of mammalian     mucin-type O-glycosylation sites. Glycobiology 15:153-164. -   Kawabata R, Wada H, Isobe M, Saika T, Sato S, Uenaka A, Miyata H,     Yasuda T, Doki Y, Noguchi Y, Kumon H, Tsuji K, Iwatsuki K, Shiku H,     Ritter G, Murphy R, Hoffman E, Old L J, Monden M, Nakayama E (2007)     Antibody response against NY-ESO-1 in CHP-NY-ESO-1 vaccinated     patients. Int J Cancer 120:2178-2184. -   Li J, Sullivan C A, Harris L (2009) Where do we place PankoMab in     the reagents used to study the MUC1 superfamily? Onkologie     32:235-237 -   Liu W L, Zhang G, Wang J Y, Cao J Y, Guo X Z, Xu L H, Li M Z, Song L     B, Huang W L, Zeng M S (2008) Proteomics-based identification of     autoantibody against CDC25B as a novel serum marker in esophageal     squamous cell carcinoma. Biochem Biophys Res Commun 375:440-445. -   Lu H, Goodell V, Disis M L (2008) Humoral immunity directed against     tumor-associated antigens as potential biomarkers for the early     diagnosis of cancer. J Proteome Res 7:1388-1394. -   Lubin R, Schlichtholz B, Bengoufa D, Zalcman G, Tredaniel J, Hirsch     A, de Fromentel C C, Preudhomme C, Fenaux P, Fournier G, Mangin P,     Laurent-Puig P, Pelletier G, Schlumberger M, Desgrandchamps F, Le     Duc A, Peyrat J P, Janin N, Bressac B, Soussi T, (1993) Analysis of     p53 antibodies in patients with various cancers define B-cell     epitopes of human p53: distribution on primary structure and     exposure on protein surface. Cancer Res 53:5872-5876. -   Mintz P J, Kim J, Do K A, Wang X, Zinner R G, Cristofanilli M, Arap     M A, Hong W K, Troncoso P, Logothetis C J, Pasqualini R, Arap     W (2003) Fingerprinting the circulating repertoire of antibodies     from cancer patients. Nat Biotechnol 21:57-63. -   Pereira-Faca S R, Kuick R, Purays E, Zhang Q, Krasnoselsky A L,     Phanstiel D, Qiu J, Misek D E, Hinderer R, Tammemagi M, Landi M T,     Caporaso N, Pfeiffer R, Edelstein C, Goodman G, Barnett M,     Thornquist M, Brenner D, Hanash S M (2007) Identification of 14-3-3     theta as an antigen that induces a humoral response in lung cancer.     Cancer Res 67:12000-12006. -   Ramachandran N, Raphael J V, Hainsworth E, Demirkan G, Fuentes M G,     Rolfs A, Hu Y, Labaer J (2008) Next-generation high-density     self-assembling functional protein arrays. Nat Methods 5:535-538. -   Rasmussen N, Ditzel H J (2009) Identification of the specificity of     isolated phage display single-chain antibodies using yeast     two-hybrid screens. Methods Mol Biol 562:165-176. -   Rauschert N, Brandlein S, Holzinger E, Hensel F, Muller-Hermelink H     K, Vollmers H P (2008) A new tumor-specific variant of GRP78 as     target for antibody-based therapy. Lab Invest 88:375-386. -   Reis C A, Sorensen T, Mandel U, David L, Mirgorodskaya E, Roepstorff     P, Kihlberg J, Hansen J E, Clausen H (1998) Development and     characterization of an antibody directed to an     alpha-N-acetyl-D-galactosamine glycosylated MUC2 peptide. Glycoconj     J 15:51-62. -   Sabbatini P J, Ragupathi G, Hood C, Aghajanian C A, Juretzka M,     Iasonos A, Hensley M L, Spassova M K, Ouerfelli O, Spriggs D R, Tew     W P, Konner J, Clausen H, Abu R N, Dansihefsky S J, Livingston P     O (2007) Pilot study of a heptavalent vaccine-keyhole limpet     hemocyanin conjugate plus QS21 in patients with epithelial ovarian,     fallopian tube, or peritoneal cancer. Clin Cancer Res 13:4170-4177. -   Sahin U, Tureci O, Schmitt H, Cochlovius B, Johannes T, Schmits R,     Stenner F, Luo G, Schobert I, Pfreundschuh M (1995) Human neoplasms     elicit multiple specific immune responses in the autologous host.     Proc Natl Acad Sci USA 92:11810-11813. -   Schietinger A, Philip M, Yoshida B A, Azadi P, Liu H, Meredith S C,     Schreiber H (2006) A mutant chaperone converts a wild-type protein     into a tumor-specific antigen. Science 314:304-308. -   Snijdewint F G M, Mensdorff-Pouilly S, Karuntu-Wanamarta A H,     Verstraeten A A, Zanten-Przybysz I, Hummel P, Nijman H W, Kenemans     P, Hilgers J (1999) Cellular and humoral immune responses to MUC1     mucin and tandem-repeat peptides in ovarian cancer patients and     controls. Cancer Immunology Immunotherapy 48:47-55.

Sorensen A L, Reis C A, Tarp M A, Mandel U, Ramachandran K, Sankaranarayanan V, Schwientek T, Graham R, Taylor-Papadimitriou J, Hollingsworth M A, Burchell J, Clausen H (2006) Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology 16:96-107.

-   Springer G F (1984) T and Tn, General Carcinoma Auto-Antigens.     Science 224:1198-1206. -   Springer G F, Tegtmeyer H (1980) On the origin of     anti-Thomsen-Friedenreich (T) antibodies. Naturwissenschaften     67:317-318. -   Stockert E, Jager E, Chen Y T, Scanlan M J, Gout I, Karbach J, Arand     M, Knuth A, Old L J (1998) A survey of the humoral immune response     of cancer patients to a panel of human tumor antigens. J Exp Med     187:1349-1354. -   Takeuchi H, Kato K, Denda-Nagai K, Hanisch F G, Clausen H, Irimura     T (2002) The epitope recognized by the unique anti-MUC1 monoclonal     antibody MY.1E12 involves sialyl alpha 2-3galactosyl beta     1-3N-acetylgalactosaminide linked to a distinct threonine residue in     the MUC1 tandem repeat. Journal of Immunological Methods     270:199-209. -   Tarp M A, Clausen H (2008) Mucin-type O-glycosylation and its     potential use in drug and vaccine development. Biochim Biophys Acta     1780:546-563. -   Tarp M A, Sorensen A L, Mandel U, Paulsen H, Burchell J,     Taylor-Papadimitriou J, Clausen H (2007) Identification of a novel     cancer-specific immunodominant glycopeptide epitope in the MUC1     tandem repeat. Glycobiology 17:197-209. -   Vollmers H P, Brandlein S (2009) Natural antibodies and cancer. N     Biotechnol 25:294-298. -   White T, Bennett E P, Takio K, Sorensen T, Bonding N, Clausen     H (1995) Purification and cDNA cloning of a human     UDP-N-acetyl-alpha-D-galactosamine:polypeptide     N-acetylgalactosaminyltransferase. J Biol Chem 270:24156-24165. 

What is claimed is:
 1. A method for identifying glycopeptides reactive with cancer-associated auto-antibodies, the method comprising: providing a panel comprising peptides immobilized on said panel, at least a plurality of said peptides comprising one or more sites amenable to glycosylation, wherein at least one of said glycosylation sites is modified with a glycan to form a glycopeptide having a peptide portion and a glycan portion; contacting said panel with an antibody-containing sample from a patient with cancer; and identifying glycopeptides in said panel that (i) are selectively recognized by antibodies in said sample, but not by antibodies in a control sample and (ii) are recognized by such antibodies in said sample through recognition of both the peptide portion and the glycan portion and not through recognition of either the peptide or the glycan alone.
 2. The method of claim 1, wherein said panel comprises peptides having an amino acid sequence comprising at least one serine or threonine residue, wherein said residue is a glycosylation site.
 3. The method of claim 1, wherein said control sample contains pooled samples from a plurality of control individuals.
 4. The method of claim 1, wherein said panel comprises mutants of said peptides.
 5. The method of claim 1, further comprising identifying minimum cancer-associated glycopeptide epitopes.
 6. The method of claim 5, further comprising elucidating the epitope structure of said identified glycopeptide epitopes.
 7. The method of claim 1, wherein each of said peptides in said panel is about 2 to about 50 amino acid residues in length.
 8. The method of claim 7, wherein each of said peptides in said panel is about 4 to about 25 amino acid residues in length.
 9. The method of claim 1, wherein said amino acid sequence of said peptides is a fragment found in a protein or variant of said protein or a conservative mutant.
 10. The method of claim 1, wherein said identified glycopeptides are found in at least one glycoprotein that is aberrantly glycosylated in cancer cells.
 11. The method of claim 1, wherein said identified glycopeptides are found in at least one glycoprotein that is overexpressed in cancer cells.
 12. The method of claim 1, wherein the serine or threonine residue is placed at about the center of each of said amino acid sequences.
 13. The method of claim 1, wherein said glycopeptides are synthesized synthetically or chemoenzymatically.
 14. The method of claim 1, wherein said glycopeptides of said panel are partially glycosylated peptides.
 15. The method of claim 1 or 14, wherein said glycopeptides of said panel are glycosylated in situ, in solution, or in vivo by recombinant expression in a host cell.
 16. The method of claim 1, wherein said glycopeptides of said panel are treated with one or more exoglycosidases to expose O-glycans.
 17. The method of claim 1, wherein said glycan is an O-glycan.
 18. The method of claim 17, wherein said O-glycan is a member selected from the group consisting of: Tn, STn, T, Truncated C3, Truncated C2, Truncated C4, non-capped type1-C3, non-capped type2-C2, non-capped type2-C4, GalNAca-3Tn, SA-type1-C3, SLea-C3, LacDiNAc-C3, LacDiNAc-C2, and LacDiNAc-C4.
 19. The method of claim 1, wherein said panel is a microarray.
 20. The method of claim 1, wherein said identified glycopeptide is selectively recognized by a cancer-associated IgG antibody.
 21. The method of claim 1, wherein said panel comprises one or more glycopeptides having an amino acid sequence selected from the group consisting of: SHHSDESDELVTDFPTDLPA (SEQ ID NO: 15); TPTPKEKPEAGTYSVNNGND (SEQ ID NO: 36); SESFPHPGFNMSLLENHTRQ (SEQ ID NO: 49); LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTCDDPRFQA (SEQ ID NO: 109); PGTSTTPSQPNSAGVQDTEM (SEQ ID NO: 116); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132); HDVETQFNQYKTEAASRYNL (SEQ ID NO: 134); ASRYNLTISDVSVSDVPFPF (SEQ ID NO: 135); VPVTRPALGSTTPPAHDVTS (SEQ ID NO: 145); and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146).
 22. A pharmaceutical composition for eliciting an immune response in a patient, comprising one or more isolated glycopeptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 86, 109, 116, 132, 134, 135 and 146, and a pharmaceutically acceptable carrier.
 23. The pharmaceutical composition of claim 22, wherein the one or more isolated glycopeptides are selected from the group consisting of LAKMYYSAVEPTKDIFTGLI (SEQ ID NO: 86); TDCGGPKDHPLTDPRFQA (SEQ ID NO: 109); TKTDASSTHHSTVPPLTSSN (SEQ ID NO: 132), and SLASQATDTFSTVPPTPPSI (SEQ ID NO: 146).
 24. A pharmaceutical composition for eliciting an immune response in a patient, comprising one or more isolated glycopeptides consisting of an epitope of 5 to 10 amino acid residues contained in a glycopeptide selected from the group consisting of SEQ ID NOs: 86, 109, 116, 132, 134, 135 and 146, and a pharmaceutically acceptable carrier, said epitope having been previously determined (i) to be selectively recognized by a subset of antibodies in sera from cancer patients, which subset recognizes neither (a) the corresponding naked peptides of said panel when not glycosylated; nor (b) the corresponding glycan when not bound to said peptide; and (ii) not to be recognized by antibodies in control sera.
 25. A panel of glycopeptides comprising at least a plurality of isolated glycopeptides immobilized on said panel, each isolated glycopeptide comprising a glycopeptide epitope, said epitope having been previously determined (i) to be selectively recognized by a subset of antibodies in sera from cancer patients, which subset recognizes neither (a) the corresponding naked peptides of said panel when not glycosylated; nor (b) the corresponding glycan when not bound to said peptide; and (ii) not to be recognized by antibodies in control sera, said plurality comprising at least 8 isolated glycopeptides, wherein each epitope is 5 to 10 amino acid residues contained in one of the amino acid sequences of SEQ ID NOs: 86, 109, 116, 132, 134, 135, 145, and 146, respectively. 